WO2022202728A1 - Primer pair, method for determining base sequence variation, and kit for determining variation in base sequence - Google Patents

Primer pair, method for determining base sequence variation, and kit for determining variation in base sequence Download PDF

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WO2022202728A1
WO2022202728A1 PCT/JP2022/012939 JP2022012939W WO2022202728A1 WO 2022202728 A1 WO2022202728 A1 WO 2022202728A1 JP 2022012939 W JP2022012939 W JP 2022012939W WO 2022202728 A1 WO2022202728 A1 WO 2022202728A1
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strand
mutation
primer
base
type
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French (fr)
Japanese (ja)
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幸信 林田
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富士フイルム株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention relates to a primer pair, a method for determining nucleotide sequence mutation, and a kit for determining nucleotide sequence mutation.
  • Gene mutation may be involved in one of the causes of individual differences in disease susceptibility, drug efficacy, side effect susceptibility, and the like. Therefore, research is underway to search for a treatment method suitable for the constitution, focusing on the difference in this gene arrangement. Gene mutations can also serve as genetic markers for many diseases. That is, elucidation of gene mutation is clinically important, and therefore establishment of an analytical method capable of detecting gene mutation is desired.
  • nucleic acid sequencing includes, for example, nucleic acid sequencing, RFLP (restriction enzyme cleavage polymorphism), ASP (allele-specific primer), ASO (allele-specific oligoprobe), single base A decompression method and the like are known.
  • RFLP restriction enzyme cleavage polymorphism
  • ASP allele-specific primer
  • ASO allele-specific oligoprobe
  • PCR polymerase chain reaction
  • Patent Document 1 Also known is a method of detecting gene mutation by performing PCR using a forward primer in which some or all of the phosphodiester bonds of nucleotides are phosphorothioated.
  • Patent Document 1 ⁇ a primer containing one or more chemically modified nucleotides, bases, or phosphodiester bonds so that the nucleotide chain extending from the primer is substantially resistant to exonuclease activity,'' Using "a primer comprising a nucleotide sequence having a sense sequence immobilized on a solid support and a sequence complementary thereto", a step of amplifying a target nucleotide sequence to generate an amplification product, based on the result Methods are disclosed for detecting and determining whether a target nucleotide is homozygous or heterozygous.
  • the present invention has been made in view of the above circumstances, and provides a primer pair used for accurate determination of genetic mutation, an accurate method for determining genetic mutation, and a nucleotide sequence mutation used for accurate determination of genetic mutation.
  • the object is to provide a determination kit.
  • both the forward primer and the reverse primer have phosphorothioated phosphodiester bonds on the 3' side of 1 to 4 consecutive nucleotides upstream from the second base from the 3' end.
  • the inventors found that the correct base sequence can be detected and determined, and have completed the present invention.
  • the present invention typically includes the following inventions.
  • a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is phosphorothioated (S)
  • S phosphorothioated
  • a primer pair with a reverse primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base toward the 5' side is S-converted.
  • Primer pair M A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side.
  • a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-formed
  • a primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of a reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed
  • Primer pair W A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-type second
  • primer pair M The primer pair according to [2] above, wherein the mutation in the base sequence of the primer pair M is a base substitution or insertion, and the primer pair M is as follows: A primer that anneals to the second strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the second strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, and has 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side.
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side.
  • primer pair M is as follows: The primer pair according to claim 2, wherein the mutation in the nucleotide sequence of the primer pair M is a deletion of a base, and the primer pair M is: A primer that anneals to the second strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5'-side nucleotide sequence adjacent to the deletion of the second strand to the 3' end.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, and extends from the second base from the 3' end to the 5' side a forward primer in which the phosphodiester bond on the 3′ side of consecutive 1 to 4 nucleotides is S-formed;
  • primer pair W is: A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to the region containing the base sequence in the wild-type second strand, and placing a complementary sequence of the base sequence of the wild-type second strand corresponding to the mutation of the mutant second strand on the 3' end side and the other nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the nucleotide sequence of the wild-type second strand corresponding to the mutation of the mutant second strand, a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 'end toward the 5' side is S-formed; A primer that anneals to the other strand (wild-type second strand) and correspond
  • a pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted.
  • Any of the above [1] to [5], wherein the phosphodiester bond on the 3' side of three consecutive nucleotides from the 2nd base from the 3' end of the primer toward the 5' side is S-ified.
  • nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair according to any one of [1] to [7] above to detect the reaction product, and the resulting detection results are also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid.
  • a nucleic acid amplification reaction is performed using the primer pair according to any one of the above [2] to [7], and the reaction product is detected. Based on the detection results obtained, the mutation in the base sequence of the nucleic acid is determined.
  • the determination method includes the following steps: (1) Using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M according to any one of [2] to [4] and [6] to [7] above, detecting the product; (2) Using the nucleic acid of the same test sample as that used in the step (1) above as a template, and using the primer pair W according to any one of [2], [5] to [7] above. a step of performing a nucleic acid amplification reaction and detecting a reaction product; (3) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (1) and (2).
  • a nucleotide sequence mutation determination kit comprising the primer pair according to any one of [1] to [7] above.
  • nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made. According to the method for determining nucleotide sequence mutations of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made. According to the method for determining nucleotide sequence mutations using the kit of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of non-S-conjugated primers obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers obtained in Example 1, in which one nucleotide is S-converted.
  • 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which two bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 3 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 4 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 4 bases are S-modified obtained in Example 1.
  • FIG. 1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers having 5 nucleotides in S, obtained in Example 1.
  • FIG. Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, targeting the PiS mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 2.
  • curve. Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, targeting the PiZ mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 3. curve.
  • FIG. 4 is a model of amplification curves of wild-type, homozygous, or heterozygous PiS mutations obtained in Example 4.
  • FIG. 6 is a model of amplification curves for wild-type, homozygous, or heterozygous PiZ mutations obtained in Example 5.
  • FIG. Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in the genomic DNA derived from the oral swab obtained in Example 6 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in saliva-derived genomic DNA obtained in Example 6 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in the genomic DNA derived from the oral swab obtained in Example 7 as the detection target.
  • Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in saliva-derived genomic DNA obtained in Example 7 as a detection target.
  • FIG. 2 shows an amplification curve obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting gene mutations.
  • the PKD1 gene obtained in Example 8 was confirmed to be wild-type and the PKLR gene was of a mutant type.
  • Fig. 2 shows amplification curves obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting mutations in the PKD1 gene and the PKLR gene in genomic DNA.
  • Fig. 10 is an amplification curve obtained by real-time PCR using the primer pair for VPS13B detection of the present invention, with the VPS13B mutation in canine genomic DNA obtained in Example 9 as the detection target.
  • 10 shows an amplification curve obtained by real-time PCR using the SN501Y mutation detection primer pair of the present invention, with the SN501Y mutation of the SARS-CoV-2 virus-derived genomic RNA obtained in Example 10 as the detection target.
  • 10 shows an amplification curve obtained by real-time PCR using the E484K mutation detection primer pair of the present invention, with the SARS-CoV-2 virus-derived genomic RNA and the E484K mutation as detection targets, obtained in Example 10.
  • a nucleotide sequence in which no nucleotide sequence mutation occurs is referred to as a "wild-type nucleotide sequence" with respect to the target gene whose nucleotide sequence mutation is to be detected.
  • the term “mutation” refers to a base sequence different from that of the wild type of the target gene whose base sequence mutation is to be detected, or having the above-mentioned base sequence. Examples of “mutation” include base substitution, deletion, insertion, and the like with respect to the wild-type base sequence of the gene whose base sequence mutation is to be detected.
  • the number of mutated bases is not particularly limited. 1 to 4 base mutations, preferably 1 to 3 base mutations, more preferably 1 mutation are included per mutation.
  • the base sequence of the gene to be detected when the base sequence of the gene to be detected does not have mutations, the base sequence is said to be wild-type.
  • the nucleotide sequence of the gene to be detected has mutations, the nucleotide sequence is said to be of a mutant type.
  • genotypes are determined in the present invention
  • a case where both base sequences of alleles are wild type is referred to as wild type.
  • Heterozygosity is when one of the alleles contains the mutation.
  • homozygous when both nucleotide sequences of alleles contain the same mutation, it is called homozygous. "Mutation" in the present invention includes this heterozygous and homozygous genotype.
  • PKD1 c. 9864C>A
  • PKLR c. 693 + 304G>A
  • VPS13B g.4411950_4411953delGTTT
  • MFSD8 HEXB
  • GLB1 etc.
  • viruses for example, SARS-CoV-2, N501Y (A23063T), E484K ( G23012A) and the like.
  • target genes for detecting and determining nucleotide sequence mutations include, for example, BRAF, Pis, PiZ, etc. are mentioned.
  • complementary base refers to a base that is complementary to a certain base.
  • complementary strand refers to a nucleic acid strand having a base sequence complementary to a given nucleic acid strand.
  • complementary sequence refers to a base sequence that is complementary to a given base sequence.
  • nucleotide sequence includes both a single base sequence and a multiple base sequence.
  • FIG. S1 shows a mutation in which one strand of a wild-type double-stranded nucleic acid and one strand of a mutant double-stranded nucleic acid are substituted for a base sequence X with a base sequence Y for a gene.
  • Figure 10 illustrates a chain with
  • mutant sequence represents the base sequence of Y of the nucleic acid with mutation.
  • nucleotide sequence corresponding to the mutation refers to the X nucleotide sequence of the wild-type nucleic acid.
  • the term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
  • a “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing X in a wild-type nucleic acid.
  • FIG. S2 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the base sequence Y is between the base sequence X1 and the base sequence X2.
  • mutant sequence represents the base sequence of Y of the nucleic acid with mutation.
  • nucleotide sequence corresponding to the mutation represents the nucleotide sequence of X1 and/or X2 of the wild-type nucleic acid.
  • the term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
  • a region containing a (wild-type) base sequence corresponding to a mutation refers to a base sequence region (+) containing X1 and X2 of a wild-type nucleic acid.
  • FIG. S3 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the nucleotide sequence Y between the nucleotide sequence X1 and the nucleotide sequence X2 is It is a diagram illustrating the case of having a missing mutation.
  • the “mutant sequence” represents the consecutive base sequences of X1 and X2.
  • Wild-type nucleotide sequence corresponding to mutation refers to the Y nucleotide sequence of the wild-type nucleic acid.
  • mutant-containing region refers to a region (*) of a base sequence containing a mutant sequence in a nucleic acid chain having a mutation.
  • a “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing Y in a wild-type nucleic acid.
  • Nucleic acids include DNA and RNA, preferably DNA.
  • the abbreviations commonly used in the field of the present invention (adenine “A” or “a”, guanine “G” or “g”, cytosine “C” or “c”) ”, and thymine as “T” or “t”).
  • Primer pair of the present invention is "A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is phosphorothioated (S-formed), and the 3' end A pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base to the 5' side is S-converted".
  • Forward primer is hereinafter referred to as "F primer”.
  • reverse primer is described as “R primer”.
  • the F primer and R primer according to the primer pair of the present invention are composed of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side of the phosphodiester bond moiety on the 3' side.
  • One of the two oxygen atoms of the residue is substituted with a sulfur atom, so-called phosphorothioated (also described herein as “S”).
  • the number of S-modified nucleotides in the primer according to the present invention is preferably 1 to 4, more preferably 2 to 4, and particularly preferably 3.
  • the number of S-nucleotides in the F primer and the number of S-nucleotides in the R primer may be the same or different, but are preferably the same.
  • the length of the primer according to the present invention is preferably 10 bases or more, which is considered to be the number of bases required to maintain specificity as a primer sequence, and more preferably 20 bases or more. Examples include 20 to 60 bases.
  • a well-known method may be used to synthesize an S-primer into which a phosphorothioate bond has been introduced.
  • a phosphorothioate bond is added to the required site by a known method. should be introduced.
  • a phosphorothioate bond is introduced into the oligonucleotide instead of a normal phosphodiester bond by performing an oxidation treatment with an appropriate S-conjugation reagent (phosphorothioate reagent). can be done.
  • Beaucage's reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide), TETD/Acetonitrile (TETD: tetraethylthiuram disulfide), and the like are known as sulfide reagents.
  • S-primers are also available through the custom service of vendors.
  • Preferred primer pairs of the present invention include, for example, primer pair M and primer pair W below.
  • Primer pair M A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side.
  • an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides from the second base from the 3′ end toward the 5′ side is S-converted;
  • a primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted,
  • Primer pair W A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-
  • the "double-stranded nucleic acid" in primer pair M and primer pair W of the present invention includes DNA. Also included are RNA and its complementary strand, and cDNA obtained by reverse transcription using RNA as a template and its complementary strand.
  • one strand (second strand) of a double-stranded nucleic acid having a nucleotide sequence mutation and "one strand (second strand) of a wild-type double-stranded nucleic acid )”, the description of “one strand (second strand)”, and “the other strand of the double-stranded nucleic acid having a mutation (first strand)” and “the other side of the wild-type double-stranded nucleic acid
  • the description of "the other strand (first strand)” in relation to "the strand (first strand)” simply means that it refers to one strand and the other strand of a double-stranded nucleic acid, and the first Strand and secondary strand each have no particular meaning.
  • primer that anneals to one strand (second strand) of a double-stranded nucleic acid means a complementary strand (first This means that the primer has the same base sequence as the base sequence of the (strand).
  • the "primer that anneals to the other strand (first strand) of the double-stranded nucleic acid” means the complementary strand (first strand) of the region of the first strand to which the primer anneals. It means that the primer has the same base sequence as the base sequence of the second strand).
  • the "3' terminal side" refers to a region containing several nucleotides including the 3' terminal nucleotide, preferably a region containing 1 to 4 nucleotides.
  • the F primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3′ terminal nucleotide of the base sequence. It preferably has a sequence complementary to the mutated sequence of the second strand of the mutated double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the mutated base of the second strand.
  • the R primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3' terminal nucleotide.
  • it has the complementary sequence of the mutant sequence of the strand.
  • the base of the 3' terminal nucleotide is the complementary base of the mutated base of the first strand.
  • nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the second strand of the wild-type double-stranded nucleic acid that corresponds to the mutation in the second strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the second strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
  • nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation in the first strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
  • the above-described primer pair M of the present invention provides an amplification product in a nucleic acid amplification reaction using this primer pair when the nucleotide sequence of the nucleic acid of the test sample has a mutation.
  • a primer pair that does not have a mutation does not yield an amplification product in a nucleic acid amplification reaction using this primer pair. That is, the primer pair M of the present invention is a primer pair for mutation detection.
  • the primer pair W of the present invention is a primer pair that gives an amplification product in a nucleic acid amplification reaction using this primer pair when the base sequence of the nucleic acid in the test sample is of the wild type. That is, the primer pair W of the present invention is a wild-type detection primer pair that is wild-type with respect to the mutation to be detected.
  • primer pair M and primer pair W of the present invention will be described in detail below.
  • primer pair M of the present invention examples include a primer pair (M-1) for detecting nucleotide sequence substitution or insertion mutations, or a primer pair (M-2) for detecting nucleotide deletion mutations.
  • Primer pair (M-1) of the present invention for detecting nucleotide sequence substitution or insertion is as follows.
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side
  • the other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the mutation of the second strand, and is continuous from the second base from the 3'-end toward the 5'-side.
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side.
  • a primer pair of R primers in which the phosphodiester bond on the 3' side of the nucleotide is S-sylated.
  • Substitution or insertion mutations in the base sequence to be detected are preferably substitutions or insertions of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide of the second strand It is the same as the complementary sequence of the mutant sequence, and the other base sequence is the same as the complementary sequence of the 3' side base sequence adjacent to the mutation in the second strand, from the second base from the 3' end an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides toward the 5′ side is S-formed;
  • a primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide are the mutation of the first strand It is the same as the complementary sequence of the sequence, and the rest of the nu
  • a primer that anneals to the second strand of a double-stranded nucleic acid having a single-base substitution or insertion mutation anneals to the region containing the mutation, and has a 3′ terminal nucleotide that is complementary to the mutated base of the second strand.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, from the second base from the 3' end to the 5' side
  • An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed
  • the other nucleotide sequence is the same as the complementary sequence of the 3′ side nucleotide sequence adjacent to the mutation of the first strand, and is continuous from the second base from the 3′ end toward the 5′ side.
  • a primer pair of R primers in which the phosphodiester bonds on the 3′ sides of the four nucleotides are S-sylated.
  • FIG. A illustrates the case where the number of S-nucleotides in each primer is three.
  • FIG. A(1) A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
  • Figure A (2) Primer pair for detecting single base substitution
  • Figure A (2) shows that the bases marked ⁇ in the wild-type base sequence are substituted from guanine (g) to adenine (a) in the first strand.
  • An example of a primer pair (M-1) for detecting a single-base substitution mutation in which cytosine (c) is substituted with thymine (t) in the second strand is shown.
  • the F primer has at its 3′ end a, the complementary base of the mutated base (t) of the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the mutated nucleotide t of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed.
  • the R primer has t at its 3' end, which is the complementary base of the mutated base (a) of the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide a of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure A (3) Primer pair for detecting double-base substitution
  • An example of a primer pair (M-1) that detects a 2-base mutation in which the chain is substituted from ca to tg is shown.
  • the F primer has at its 3' end ca, the complementary sequence of the mutated sequence (tg) of the second strand.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattgg) of the 3'-side nucleotide sequence (ccaatcaca) adjacent to the mutated sequence tg of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgg) from the second base from the 3' end of the F primer toward the 5' side (cgg) is S-formed.
  • the R primer has at its 3' end tg, the complementary sequence of the mutant sequence (ca) of the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to ca, which is the mutant sequence of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
  • An example of a primer pair (M-1) that detects a 3-base substitution mutation in which cac is replaced with tga in the strand is shown.
  • the F primer has at its 3' end tca, the complement of the second strand mutant sequence (tga).
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattg) of the 3′-side nucleotide sequence (caatcaca) adjacent to the mutated sequence tga of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed.
  • the R primer has at its 3' end tga, the complementary sequence of the first strand mutant sequence (tca).
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated sequence tca of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. B exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. B(1) A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
  • Figure B (2) Primer pair for detecting single base insertion Figure B (2) shows that t is inserted at the position marked ⁇ in the first strand and marked ⁇ in the second strand against the wild-type base sequence.
  • An example of a primer pair (M-1) for detecting a single-base insertion mutation in which a is inserted at the position of is shown.
  • the F primer has t at its 3' end, which is the complementary base of base (a) inserted in the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the 3'-side nucleotide sequence (caccaatcaca) adjacent to the mutated nucleotide a of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the F primer toward the 5' side is S-formed.
  • the R primer has at its 3' end a, the complementary base of the base (t) inserted in the first strand.
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide t of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure B (3) Primer pair for detecting double base insertion Figure B (3) shows that ta is inserted at the position marked ⁇ in the first strand and marked ⁇ in the second strand against the wild-type base sequence.
  • An example of a primer pair (M-1) for detecting a 2-base insertion mutation in which ta is inserted at the position of is shown.
  • the F primer has at its 3' end ta, the complement of ta inserted in the second strand.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the mutant sequence ta of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the F primer toward the 5' side (tgt) is S-formed.
  • the R primer has at its 3' end ta, the complementary sequence of ta inserted in the first strand.
  • the other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutant sequence ta of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
  • Figure B (4) Primer pair for detecting 3-base insertion
  • a tag is inserted at the position marked ⁇ in the first strand against the wild-type base sequence, and marked ⁇ in the second strand.
  • An example of a primer pair (M-1) for detecting a 3-base insertion mutation in which cta is inserted at the position of is shown.
  • the F primer has tag at its 3' end, which is the complementary sequence of cta inserted in the second strand.
  • the other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the mutant sequence cta of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (atg) from the second base from the 3' end of the F primer toward the 5' side (atg) is S-formed.
  • the R primer has cta at its 3' end, which is the complementary sequence of the tag inserted in the first strand.
  • the other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to tag, which is the mutant sequence of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tcg) from the second base from the 3' end of the R primer toward the 5' side (tcg) is S-formed. .
  • primer pair M-1 of the present invention for example, PiS mutation, which is a single base substitution mutation of human ⁇ 1-antitrypsin gene (genomic DNA GenBank Accession No. NG_008290.1, mRNA GenBank Accession No. NM_000295.5)
  • the mutation detection primer pair (primer pair M-1 of the present invention, corresponding to the case of FIG. A(2) above) will be described below as an example.
  • PiS mutation which is a gene mutation of human ⁇ 1-antitrypsin, is the following single base substitution.
  • the human PiS mutation is a single-base substitution in which the 863rd adenine (A) in the coding DNA is replaced with thymine (T), and as a result, the 288th Glu in the ⁇ 1-antitrypsin amino acid sequence is replaced with Val. Mutation.
  • the primer pair of the present invention for detecting this single base substitution, the strand in which A is replaced with T in the double-stranded DNA of the ⁇ 1-antitrypsin gene is defined as the first strand.
  • the strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
  • the * mark indicates the position where the phosphodiester bond is S-converted.
  • PiS mutation detection primer pair (M-1) for example, the following are designed.
  • the F primer of the PiS mutation detection primer pair has T at the 3′ end, which is the complementary base of the mutated base (A) of the second strand of the double-stranded nucleic acid having the PiS mutation.
  • Other nucleotide sequences are designed to be the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the mutated nucleotide (A) of the second strand.
  • the R primer of the pair of primers for detecting PiS mutation type has, at its 3' end, A, which is the complementary base to the base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation.
  • nucleotide sequences are designed to be the same as the 3' nucleotide sequence adjacent to the mutated nucleotide (T) of the first strand. Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
  • the F primer has the same 3′ terminal base as the mutated base (T) of the PiS gene, and the other base sequence is 5 from the mutated base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation. It is designed to be the same as the base sequence facing the ' side.
  • the R primer has the same 3′ terminal base as the complementary base (A) of the mutated base (T) of the PiS gene, and the other base sequence is the same as the first strand of the double-stranded nucleic acid having the PiS mutation.
  • the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
  • primer pair M of the present invention include the following mutation detection for detecting PiZ mutation (single nucleotide mutation, c.1096G>A p.Glu366Lys), which is another genetic mutation of human ⁇ 1-antitrypsin. Primer pairs are included.
  • primer pair M-1 of the present invention include those listed in Table 1 below.
  • Primer pair (M-2) of the present invention for detecting nucleotide sequence defects is as follows. A primer that anneals to the second strand of a double-stranded nucleic acid having a deletion mutation, anneals to the region containing the deletion, and anneals to the 5'-side nucleotide sequence adjacent to the deletion of the second strand.
  • the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, from the second base from the 3' end toward the 5' side
  • An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed A primer that anneals to the first strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5' base sequence adjacent to the deletion of the first strand to the 3' end.
  • the other base sequence is the same as the complementary sequence of the salt sequence on the 3' side adjacent to the deletion of the first strand, from the second base from the 3' end toward the 5' side
  • a pair of R primers in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-converted.
  • “Annealing to the deletion-containing region" of the primer pair (M-2) of the present invention means binding to at least bases on both ends (5' side and 3' side) of the deletion position.
  • Deletion mutations in the base sequence to be detected are preferably deletions of 1 to 4 bases. A deletion of 1 to 3 bases is more preferred. A single base deletion is more preferred.
  • FIG. C shows three examples of primer pairs (M-2) that detect mutations in which one base is deleted at the position of the arrow in the wild-type base sequence.
  • the first strand lacks the arrow g and the second strand lacks the arrow c.
  • blanks in the base sequences indicate that bases are missing, and in fact, the 5' and 3' bases of the blanks are continuous. Note that FIG. C illustrates a case where the number of S-nucleotides in each primer is three.
  • FIG. C(1) A representation of the base sequences of the wild-type first and second strands.
  • the F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed.
  • the R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • the F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed. .
  • the R primer has at its 3' end ac, the complementary sequence of the 5' nucleotide sequence (gt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
  • the F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
  • the R primer has at its 3' end acc, the complementary sequence of the 5' nucleotide sequence (ggt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cag) from the second base from the 3' end of the R primer toward the 5' side (cag) is S-formed. .
  • FIG. D shows three examples of primer pairs (M-2) for detecting mutations in which 4 bases marked with ⁇ are deleted from the wild-type base sequence.
  • the first strand lacks ggtg marked with ⁇
  • the second strand lacks cacc marked with ⁇ .
  • a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous. Note that FIG. D illustrates a case where the number of S-nucleotides in each primer is three.
  • FIG. D(1) A representation of the base sequences of the wild-type first and second strands.
  • Figure D (2) Primer pair-1 for detecting 4-base deletion
  • the F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect.
  • the rest of the base sequence is the same as the complementary sequence (tgtgatt) of the base sequence (aatcaca) on the 3' side adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tta) from the second base from the 3' end of the F primer toward the 5' side (tta) is S-formed.
  • the R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • the F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3′-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctt) from the second base from the 3' end of the F primer toward the 5' side (ctt) is S-formed. .
  • the R primer has at its 3' end aa, which is the complementary sequence of the 5' nucleotide sequence (tt) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
  • Figure D Primer pair for detecting 4-base deletion -3
  • the F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3'-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
  • the R primer has at its 3' end aat, which is the complementary sequence of the 5' nucleotide sequence (att) adjacent to the first strand defect.
  • the rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (aag) from the second base from the 3' end of the R primer toward the 5' side (aag) is S-formed. .
  • a primer pair for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method.
  • primer pair M-2 of the present invention include the following.
  • a 4-base deletion in the canine VPS13B gene is known, and the following primer pair is designed as a primer pair for detecting the mutation.
  • VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73)
  • primer pair M-2 of the present invention include those listed in Table 2 below.
  • the primer pair M of the present invention is shown in FIG. ), and the primer pair of Figure D(2) are preferred. More preferred are the primer pairs of Figure A(2) and Figure B(2).
  • primer pair W of the present invention examples include a wild-type detection primer pair (W-1) used for detecting substitution or insertion mutation of a base sequence, or a wild-type detection primer pair (W-1) used for detecting base deletion mutation.
  • Type-detecting primer pair (W-2) can be mentioned.
  • Primer pair (W-1) of the present invention is as follows.
  • the rest of the nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide sequence corresponding to the mutation of the mutant second strand.
  • an F primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed;
  • the other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation of the mutant first strand.
  • a pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucle
  • substitution or insertion mutation of the base sequence to be detected is preferably a substitution or insertion of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
  • primer pair W-1 of the present invention are preferred.
  • an F primer A primer that anneals to the first strand of the wild-type double-stranded nucleic acid (wild-type first strand), and replaces the first strand of the mutant double-stranded nucleic acid (mutant first strand) or Annealed to a region containing a nucleotide sequence in the wild-type first strand corresponding to the insertion mutation, and 1 to 4 consecutive nucleotides including the 3′ terminal nucleotide correspond to the mutation in the mutant first strand and the other nucleotide sequence is adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation in the mutant first strand.
  • wild-type first strand that anneals to the region containing the nucleotide sequence in the wild-type first strand corresponding to the substitution or insertion mutation, and the 3′ terminal nucleotide corresponds to the mutation in the mutant-type first strand and the other base sequence is complementary to the base sequence on the 3' side adjacent to the base sequence of the wild-type first strand corresponding to the mutation of the first strand of the mutant type
  • the primer pair (W-1) of the present invention which is a wild-type detection primer pair used for detecting substitution of base sequences, will be described with reference to FIG. A2 below.
  • FIG. A2 exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. A2(1) shows an example of the primer pair (W-1) of the present invention used for wild type detection when detecting a single nucleotide substitution.
  • the detection target is a single base substitution in which the wild-type base sequence is replaced with a by g in the first strand, and c is replaced by t in the second strand. indicates when The F primer is the second strand of a wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (g) of c, which is the base of the second strand of the type.
  • the other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to c, which is the nucleotide of the wild-type second strand corresponding to the mutated nucleotide (t) of the second strand of the mutant type. Same as array (tgtgattggt).
  • array (tgtgattggt) the nucleotide of the wild-type second strand corresponding to the mutated nucleotide (t) of the second strand of the mutant type.
  • array tgtgattggt
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (c) of g, which is the base of the first strand of the type).
  • the other nucleotide sequence is complementary to the nucleotide sequence (catccgc) on the 3′ side adjacent to g, which is the base of the wild-type first strand corresponding to the mutated nucleotide (a) of the first strand of the mutant type. Same as array (gcggatg).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. A2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting a two-base substitution.
  • W-1 primer pair
  • FIG. A2 shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting a two-base substitution.
  • tg is substituted with ca in the first strand
  • ca is substituted with tg in the second strand.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid ( It has a complementary sequence (tg) of ca, which is the nucleotide sequence of the wild-type second strand), at its 3' end.
  • the other nucleotide sequence is the 3′-side nucleotide sequence (ccaatcaca) adjacent to ca, which is the nucleotide sequence of the wild-type second strand corresponding to the mutant nucleotide sequence (tg) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattgg) of
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated base sequence (ca) of the first strand of the mutant double-stranded nucleic acid having the above two base substitutions (mutant first strand). It has a complementary sequence (ca) of tg, which is the base sequence of (wild-type first strand), at its 3' end.
  • the other base sequence is the base sequence (catccgc) on the 3' side adjacent to tg, which is the base sequence of the wild-type first strand corresponding to the mutant base sequence (ca) of the first strand of the mutant type.
  • FIG. A2(3) shows an example of the primer pair (W-1) of the present invention, which is used for wild-type detection when detecting mutations in which three bases are substituted.
  • detection target is 3 base substitutions in which gtg is substituted with tca in the first strand and cac is substituted with tga in the second strand.
  • the F primer is a wild-type double-stranded nucleic acid second strand ( It has a complementary sequence (gtg) of cac, which is the nucleotide sequence of the wild-type second strand), at its 3' end.
  • the other base sequence is the base sequence (caatcaca) on the 3' side adjacent to cac, which is the base sequence of the wild-type second strand corresponding to the mutant base sequence (tga) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattg) of
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated nucleotide sequence (tca) of the first strand of the mutant double-stranded nucleic acid having the three base substitutions (mutant first strand). It has a gtg complementary sequence (cac), which is the base sequence of (wild-type first strand), at its 3' end. The other base sequence is the base sequence (catccgc) on the 3' side adjacent to the base sequence gtg of the wild-type first strand corresponding to the mutant base sequence (tca) of the first strand of the mutant type.
  • Wild-type detection primer pair (W-1) used for insertion detection The primer pair (W-1) of the present invention, which is a wild-type detection primer pair used for detecting insertion of a nucleotide sequence will be explained based on the following diagram B2.
  • FIG. B2 exemplifies the case where the number of S-nucleotides in each primer is three.
  • FIG. B2(1) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when single-base insertion is detected.
  • FIG. B2(1) shows the case of detecting a single base insertion in which t is inserted in the first strand and a is inserted in the second strand at the positions indicated by the arrows in the wild-type base sequence.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end c, the complementary base of the 5' base (g) adjacent to the (strand) position.
  • the other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand. be.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. and the other base sequence is the 3′-side base (c) adjacent to the 3′-side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the base sequence (atccgc).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • FIG. B2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion.
  • FIG. B2(2) shows a case in which 2-base insertions in which ta is inserted in the first strand and ta is inserted in the second strand at the positions indicated by arrows in the wild-type base sequence are detected.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand).
  • the other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type second strand corresponding to the insertion mutation of the second strand of the mutant type. Same as array (tgtgattggt).
  • array (tgtgattggt) the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand).
  • the other nucleotide sequence is the complement of the nucleotide sequence (atccgc) on the 3' side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type first strand corresponding to the insertion mutation of the first strand of the mutant type. Same as array (cggatg).
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ggt) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • FIG. B2(3) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion.
  • W-1 the primer pair of the present invention used for wild-type detection when detecting double base insertion.
  • ta is inserted in the first strand at the position of the arrow, and 2 base insertions in which ta is inserted in the second strand are to be detected.
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end ca, which is a complementary sequence of gt, which is the 5' base sequence of the (strand) position.
  • the rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand.
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the F primer toward the 5' side (cgt) is S-formed. .
  • the R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. And other nucleotide sequences are the 3′ side adjacent to the 3′ side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the nucleotide sequence (atccgc). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • primer pair W-1 of the present invention for example, a wild-type detection primer pair (primer pair W-1 of the present invention, FIG. A2 This corresponds to the case of (2).) will be described below as an example.
  • PiS mutation which is a gene mutation of ⁇ 1-antitrypsin.
  • the primer pair W-1 of the present invention which is a wild-type detection primer pair used for detecting this single base substitution
  • the double-stranded DNA of the ⁇ 1-antitrypsin gene in which A was replaced with T was used. is the first strand.
  • the strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
  • the * mark indicates the position where the phosphodiester bond is S-converted.
  • PiS wild-type detection primer pair (W-1) for example, the following are designed.
  • the F primer of the wild-type detection primer pair used for PiS mutation detection is a wild-type corresponding to the mutated base (A) of the second strand of the double-stranded nucleic acid having a PiS mutation (second strand of the mutant type). It has A, which is the complementary base of the base (T) of the second strand (wild-type second strand) of the double-stranded nucleic acid, at the 3′ end.
  • Other nucleotide sequences are the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide (T) corresponding to the mutant second strand nucleotide (A).
  • the R primer of the primer pair is the first strand of a wild-type double-stranded nucleic acid ( It has a T at the 3′ end, which is the complementary base of base (A) of wild-type first strand).
  • Other base sequences are designed to be the same as the base sequence on the 3' side adjacent to the wild-type first-strand base (A) corresponding to the mutant first-strand base (T). be done.
  • the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
  • the F primer has the same 3′ terminal base as the wild-type base (A) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type double-stranded nucleic acid of the PiS gene. It is designed to have the same nucleotide sequence as the 5′ side of the wild-type nucleotide (A) corresponding to the above-mentioned mutated nucleotide (T) of the first strand.
  • the R primer has the same 3′ terminal base (A) as the complementary base (A) of the wild-type base (T) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type base (T) of the PiS gene. designed to be the same as the complementary sequence of the base sequence toward the 3′ side adjacent to the wild-type base (A) corresponding to the mutated base (T) of the first strand of the double-stranded nucleic acid of the type be.
  • the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
  • primer pair W of the present invention examples include a wild-type detection primer pair used for detecting PiZ mutation (single nucleotide mutation), which is another gene mutation of ⁇ 1-antitrypsin.
  • primer pair W-1 of the present invention include those listed in Table 3 below.
  • Primer pair (W-2) of the present invention which is a pair of primers for wild-type detection used for detecting deletion of nucleotide sequences, is as follows.
  • a primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a defect in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) A complementary sequence of the base sequence of the wild-type second strand that anneals to the region containing the base sequence in the wild-type second strand and corresponds to the deletion of the mutant-type second strand, or the wild-type first strand It has a complementary sequence of the base sequence of the second strand and the base sequence of the 5' side adjacent to it at the 3' end side, and the other base sequence is the wild-type second strand corresponding to the deletion of the second strand of the mutant type.
  • an F primer in which the phosphodiester bond on the side is S-ified A primer that anneals to the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) and corresponds to a defect in the first strand of a mutant-type double-stranded nucleic acid (mutant-type first strand)
  • the first strand of the wild type has a sequence complementary to the base sequence of the first strand and the base sequence of the 5' side adjacent thereto at the 3' end side, and the other base sequence is the first strand
  • Wild-type detection primer pair (W-2) used for detecting 1-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 1-base deletion in a nucleotide sequence ( W-2) will be explained with reference to Figure C2 below.
  • FIG. C2 shows a case where a 1-base deletion is targeted for detection, in which the first strand lacks the arrow g and the second strand lacks the arrow c in the wild-type base sequence.
  • FIG. C2 shows the case where the number of S-nucleotides in each primer is 3.
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -1
  • the F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand).
  • the other nucleotide sequence is a complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -2
  • the F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3' end a complementary sequence (ca) of the base g of the first strand) and the adjacent 5' base (t).
  • the other nucleotide sequence is a complementary sequence (gcggatgc) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the second strand of the wild type corresponding to the deletion of the first strand of the mutant type. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the R primer toward the 5' side (cgt) is S-formed. .
  • Figure C2 Primer pair for detecting 1 base deletion (wild type) -3
  • the F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end a complementary sequence (gc) of the base c of the chain) and the adjacent 5' base (g).
  • the other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand.
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand).
  • the other nucleotide sequence is the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
  • Wild-type detection primer pair (W-2) used for detecting 4-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 4-base deletion in a nucleotide sequence ( W-2) will be explained based on the following diagram D2.
  • the first strand lacks ggtg marked with ⁇
  • the second strand lacks cacc marked with ⁇ . show.
  • FIG. D2 a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous.
  • FIG. D2 shows the case where the number of S-nucleotides in each primer is three.
  • Figure D2 Primer pair for detecting 4-base deletion
  • the F primer is a wild-type double-stranded nucleic acid corresponding to the deletion of the second strand of a mutant double-stranded nucleic acid having a 4-base deletion (mutant second strand). It has at its 3′ end a complementary sequence (ggtc) of cacc, which is the base sequence of the second strand (wild-type second strand).
  • the other nucleotide sequence is a complementary sequence (tgtgatt ) is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
  • the R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end the complementary sequence (cacc) of ggtg, which is the base sequence of the ggtg chain).
  • the other nucleotide sequence is a complementary sequence (gcggatg ) is the same as
  • the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cac) from the second base from the 3' end of the R primer to the 5' side (cac) is S-formed. .
  • a primer pair W-2 for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method, although not explained using the figures.
  • primer pair W-2 of the present invention include the following.
  • the following primer pair is designed as a wild-type detection primer pair for detecting the above-described 4-base deletion in the canine VPS13B gene.
  • the primer pairs shown in Figure A2(2), Figure B2(2), Figure C2(2), and Figure D2(2) are preferable. Considering the ease of determination of nucleotide sequence mutations, the primer pairs of Figure A2(2) and Figure B2(2) are more preferable.
  • primer pair W-2 of the present invention include those listed in Table 4 below.
  • the method for determining a nucleotide sequence mutation of the present invention comprises "Using the nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair of the present invention (for example, primer pair M or/and primer pair W) to detect the reaction product, and the resulting detection result is also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid.” is.
  • Test sample used in the method for determining a nucleotide sequence mutation of the present invention includes oral swabs (oral swabs), nasal swabs, nasopharyngeal swabs, pharyngeal swabs, saliva, Body fluids such as ascites, pleural effusion, nerve root fluid, lymphatic fluid, cerebrospinal fluid, digestive fluid, intratracheal aspirate, pharyngeal mucus, sputum, transbronchial sample, bronchial lavage, various clinical materials such as pleural effusion, plasma, serum, total Blood samples such as blood, tissue sections, tissue lavage fluids, feces, urine and other biological samples, cultured cells, cell supernatants, cell lysates, or pretreated samples such as heat-treated samples and those adjusted by dilution or concentration, etc., as necessary.
  • oral swabs oral swabs
  • nasal swabs nasal swabs
  • the specimen from which the test sample according to the present invention is obtained is not limited to animals, plants, viruses, etc., as long as it contains the nucleic acid of the gene to be detected.
  • Examples of animals from which the test sample according to the present invention can be obtained include mammals (humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, bats, mice, rats, etc.), Examples include reptiles and the like.
  • Viruses from which test samples according to the present invention can be obtained include SARS-CoV-2 and the like.
  • test sample nucleic acid or “test sample-derived nucleic acid” used for detecting gene mutation according to the present invention, a nucleic acid extracted and purified from the above-described test sample, or a nucleic acid amplification detection system, etc. It may be an amplified nucleic acid. Nucleic acids may be double-stranded or single-stranded, and may be DNA or RNA. In the case of RNA, cDNA obtained by reverse transcription may be used as the nucleic acid.
  • the nucleic acid of the test sample used in the method for determining the nucleotide sequence of the present invention may be isolated and purified from the test sample by methods commonly used in this field.
  • a nucleic acid amplification reaction commonly performed in this field may be performed, except that the primer pair of the present invention is used.
  • the primer pair of the present invention it is hybridized with the nucleic acid in the sample, and nucleic acid amplification by DNA polymerase [for example, PCR (polymerase chain reaction), LAMP (Loop-mediated Isothermal Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method] to extend the primer.
  • DNA polymerase for example, PCR (polymerase chain reaction), LAMP (Loop-mediated Isothermal Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method
  • the conditions, operation method, etc. of the nucleic acid amplification reaction may be
  • the "method for detecting a reaction product obtained by a nucleic acid amplification reaction” includes, for example, the intercalator method, the TaqMan TM real-time PCR method, the MGB Eclipse Probe System method, the Molecular Beacons Probe Technology method, the LUX Fluorogenic Primer method, Quenching probe-PCR (QP) method, a method based on electrophoresis of the obtained primer extension product after performing nucleic acid amplification reaction, a method based on the result, a method obtained by performing nucleic acid amplification reaction using labeled primers
  • QP Quenching probe-PCR
  • a variety of detection methods are included, including methods that measure labeling of primer extension products.
  • real-time PCR using a known intercalator e.g., EvaGreen TM (manufactured by Biotium), SYBR TM Green I (manufactured by Molecular Probe), etc.
  • a known intercalator e.g., EvaGreen TM (manufactured by Biotium), SYBR TM Green I (manufactured by Molecular Probe), etc.
  • Fluorescence intensity derived from the intercalator is detected.
  • nucleic acid amplification reaction is performed using the purified nucleic acid derived from the test sample as a template, and the mutation to be detected in the nucleic acid is used. Amplify the region containing (1st PCR). Then, using the amplified product as a template, a nucleic acid amplification reaction using the primer pair of the present invention may be performed.
  • Method for determining mutation As a method for determining mutation, 1) A method for determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation; or 2) Determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type or mutant. how to, is mentioned. Each determination method will be described below.
  • a determination method comprising the following steps: (i) using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M of the present invention, and detecting the reaction product; (ii) a step of performing a nucleic acid amplification reaction using the primer pair W of the present invention using the nucleic acid of the same test sample as that used in step (i) above as a template, and detecting the reaction product; (iii) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (i) and (ii). ] is mentioned.
  • Primer pair M of the present invention and primer pair W of the present invention are as described in the section " ⁇ 1. Primer pair of the present invention>", and preferred examples and specific examples are also the same.
  • the method of carrying out the nucleic acid amplification reaction using each primer pair in the above steps (i) and (ii) may be carried out according to the method described in the section "(2) Nucleic acid amplification reaction", which is preferable. Examples, specific examples, etc. are the same. Either step (i) or step (ii) may be performed first, and the order of the steps does not matter.
  • Step (iii) may be performed by performing steps (i) and (ii) to obtain an amplification curve, respectively, and comparing the amplification curve with a control amplification curve obtained by the method described below. good.
  • Determining method based on amplification curve Determining whether the nucleotide sequence of the nucleic acid of the test sample is a wild type, homozygous mutation, or heterozygous mutation (that is, determining genotype)
  • a method of making determination based on an amplification curve of fluorescence intensity measured in a nucleic acid amplification reaction there is a method of making determination based on an amplification curve of fluorescence intensity measured in a nucleic acid amplification reaction.
  • a method for judging by comparing an amplification curve obtained using a test sample-derived nucleic acid as a template with a control amplification curve will be described below as an example.
  • An oligonucleotide (wild-type control) containing the nucleotide sequence corresponding to the above mutation is designed and prepared.
  • Primer pairs of the present invention> using a primer pair M for detecting a mutation to be detected and a primer pair W for wild-type detection corresponding to the same mutation to be detected, and using a control of the combination shown in Table 5 below as a template. , prepare a sample control amplification curve using the primer pairs of the invention.
  • a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 1-1'). Further, using the same wild-type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 1-2'). Then, the resulting amplification curve 1-1' and amplification curve 1-2' are plotted in one graph, and this graph is used as a wild-type control.
  • a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 2-1'). Further, using the same mutant type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 2-2'). Then, the obtained amplification curve 2-1' and amplification curve 2-2' are represented in one graph, and this graph is used as a heterozygous control.
  • a mixture of the same wild-type control and the same mutant control as above is used as a template, and the primer pair W of the present invention is used to carry out a nucleic acid amplification reaction to obtain an amplification curve (amplification curve 3-1').
  • the same nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 3-2' ).
  • the obtained amplification curve 3-1' and amplification curve 3-2' are plotted in one graph, and this graph is used as a homozygous control. Wild-type and mutant controls are equal in weight in the templating mixture.
  • the amplification curve 1-1' rises earlier than the amplification curve 1-2'. That is, the amplification curve 1-1' is detected earlier than the amplification curve 1-2'.
  • the amplification curve 2-1' has a slower rise than the amplification curve 2-2'. That is, the amplification curve 2-1' is detected later than the amplification curve 2-2'.
  • the rises of the amplification curves 3-1' and 3-2' are comparable (detected to the same extent), i.e., the amplification curves 3-1' and 3-2' are detected with similar rapidity, and the two amplification curves are close to each other.
  • amplification curve 1 Creation of an amplification curve using a nucleic acid derived from a test sample as a template
  • a nucleic acid derived from a test sample as a template Using the same primer pair W of the present invention as used, a nucleic acid amplification reaction is performed to obtain an amplification curve (amplification curve 1).
  • a nucleic acid amplification reaction is performed using the same primer pair M of the present invention as used in the above "1-a-1) Creation of amplification curve of object", An amplification curve is obtained (amplification curve 2).
  • the obtained amplification curve 1 and amplification curve 2 are shown in one graph.
  • nucleotide sequence mutation The relationship between the amplification curve 1 and the amplification curve 2 obtained using the nucleic acid derived from the test sample obtained in 1-a-2) above as a template is Compare with the wild-type control, heterozygous control, and homozygous control obtained in a-1). If the relationship between the obtained amplification curves 1 and 2 is close to that of the wild-type control, the gene to be detected in the test sample is determined to be wild-type. If the relationship between the obtained amplification curves 1 and 2 is close to that of the heterozygous control, it is determined that the gene to be detected in the test sample is heterozygous. If the relationship between the obtained amplification curves 1 and 2 is close to that of the homozygous control, it is determined that the gene to be detected in the test sample is homozygous mutation.
  • the gene to be detected in the test sample is wild
  • the gene to be detected in the test sample is a heterozygous mutation
  • the reaction product obtained in step (i) above is detected at a cycle number close to the reaction product obtained in step (ii) above, the test sample The gene to be detected is determined to be a homozygous mutation.
  • the gene to be detected in the test sample is a homozygous mutation
  • the Ct value or Tm value obtained in the detection of the step (i) above is the Ct value obtained in the detection of the step (ii)
  • the gene to be detected in the test sample is determined to be a heterozygous mutation.
  • the method of determining the genotype whether the base sequence of the nucleic acid of the test sample according to the present invention is a wild type, homozygous mutation, or heterozygous mutation, ⁇ 1- by a real-time PCR detection system using an intercalator A method for determining the genotype of PiS, which is an antitrypsin gene mutation, will be described as an example.
  • a purified DNA sample is obtained from a test sample (eg, human blood, saliva, swab, etc.) by a known method.
  • a test sample eg, human blood, saliva, swab, etc.
  • a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified DNA sample as a template (1st PCR) to amplify the region containing the PiS mutation in the purified DNA to obtain an amplified product.
  • F primer 5'-CTGCTGATGAAATACCTGGGCAATG-3' (SEQ ID NO: 25)
  • R primer 5'-GGTTGGGGAATCACCTTCTGTCTTC-3' (SEQ ID NO: 26)
  • real-time PCR is performed, for example, as follows, using the following wild-type detection primer pair and PiS mutation type detection primer pair.
  • each of the F primer and the R primer of the PiS mutation type detection primer pair or the wild type detection primer pair of the present invention an intercalator (for example, EvaGreen TM (manufactured by Biotium), SYBR TM Green I (Molecular Probe etc.), 0.2 mM dNTPs, 0.1 to 2 units (U)/mL of DNA polymerase, respectively, in a buffer or aqueous solution containing a purified DNA sample or 1st PCR amplification product (10 pg) purified from the test sample ⁇ 10 ng) to make the reaction mixture for PCR.
  • an intercalator for example, EvaGreen TM (manufactured by Biotium), SYBR TM Green I (Molecular Probe etc.
  • 0.2 mM dNTPs 0.1 to 2 units (U)/mL of DNA polymerase
  • an oligonucleotide having a partial nucleotide sequence of the ⁇ 1-antitrypsin gene containing the mutated nucleotide sequence of the PiS mutation (PiS mutant control: for example, an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 54), and ⁇ 1- Oligonucleotide having a wild-type nucleotide sequence of ⁇ 1-antitrypsin gene (PiS wild-type control, for example, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 53, including a nucleotide sequence corresponding to PiS mutation in the antitrypsin gene ).
  • PiS wild type detection primer pair SEQ ID NO: 35-SEQ ID NO: 36
  • PiS mutation detection primer pair SEQ ID NO: 37 - SEQ ID NO: 38
  • a wild-type control is used as a template, and a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve.
  • a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve.
  • the two amplification curves obtained are merged into one figure, which is the wild-type control figure.
  • a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve.
  • a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve.
  • the two amplification curves obtained are combined into one figure, which is the figure of the homozygous control.
  • a mixture of PiS wild-type control and PiS mutant control is used as a template, and a primer pair for PiS wild-type detection is used to perform a nucleic acid amplification reaction to obtain an amplification curve.
  • a primer pair for PiS wild-type detection is used to perform a nucleic acid amplification reaction to obtain an amplification curve.
  • the amplification curve obtained by carrying out nucleic acid amplification reaction using a pair of primers for detecting PiS mutation is combined into one figure, and this figure is used as a hetero This is a diagram of the mating type control.
  • the amplification curves obtained using the test sample-derived nucleic acids are compared with the heterozygous control, wild-type control, and homozygous control graphs.
  • An amplification curve obtained using a nucleic acid derived from a test sample and using a PiS wild-type detection primer pair, and an amplification curve obtained using a nucleic acid derived from a test sample and using a PiS mutation type detection primer pair is closest to that of the heterozygous control
  • the PiS gene to be detected in the test sample is determined to be heterozygous. If the relationship between the amplification curves is the closest to that of the homozygous control, the PiS gene to be detected in the test sample is determined to be homozygous mutation. If the relationship between the amplification curves is closest to that of the wild-type control, the PiS gene to be detected in the test sample is determined to be wild-type.
  • a nucleic acid amplification reaction is performed using the primer pair M of the present invention and/or the primer pair W of the present invention, and using nucleic acid derived from a test sample as a template.
  • the reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention, but the reaction product is detected using the primer pair W of the present invention. No or very little reaction product is detected in nucleic acid amplification reactions.
  • the nucleotide sequence of the gene to be detected in the test sample is of the wild type, no or almost no reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention.
  • a reaction product is detected in the nucleic acid amplification reaction using the primer pair W of the present invention.
  • real-time PCR is performed using the primer pair M of the present invention, an intercalator, and a purified DNA sample purified from a test sample as a template. Then, the fluorescence intensity derived from the intercalator that intercalates with the amplified product is measured.
  • Real-time PCR is performed in the same manner as in the case of using the primer pair M of the present invention except that the primer pair W of the present invention is used, and the fluorescence intensity derived from the intercalator is measured.
  • the method for determining whether the nucleic acid sequence of a test sample according to the present invention is wild-type or mutant the method for determining the N501Y mutation of the SARS-CoV-2 virus will be described.
  • purified total RNA is obtained from the SARS-CoV-2 virus by a known method, and using the obtained single-stranded RNA as a template, reverse transcription is performed by a conventional method to obtain cDNA.
  • a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified cDNA as a template (1st PCR) to amplify the region containing the N501Y mutation in the cDNA to obtain an amplified product.
  • F primer 5'-CTATCAGGCCGGTAGCACACCTTG-3' (SEQ ID NO: 75)
  • R primer 5'-CCACAAACAGTTGCTGGTGCATGTAG-3' (SEQ ID NO: 76)
  • real-time PCR is performed, for example, by the intercalator method, using the following N501Y wild-type detection primer pair and N501Y mutant-type detection primer pair.
  • ⁇ N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80) ⁇ N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
  • reaction products were not detected or hardly detected, but the primer pair for detecting N501Y wild type (primer pair of the present invention If a reaction product is obtained in the nucleic acid amplification reaction using pair W), it is determined that the nucleotide sequence of the N501Y gene to be detected in the test sample is of the wild type without mutation.
  • primer pair M and primer pair W are used in the method for determining mutation of the present invention
  • the F primer of primer pair M and the F primer of primer pair W to be used are "complementary sequence of the mutated nucleotide sequence of the second strand ” are preferably the same.
  • the R primer of the primer pair M and the R primer of the primer pair W to be used preferably have the same base sequence except for the sequence corresponding to the "complementary sequence of the mutated base sequence of the first strand.”
  • primer pair M and primer pair W when primer pair M and primer pair W are used, it is preferable that the number of S-nucleotides in the F primer of primer pair M and the F primer of primer pair W to be used is the same. Similarly, the number of s-nucleotides in the R primer of primer pair M and the R primer of primer pair W to be used is preferably the same.
  • the kit of the present invention is a kit for determining whether the base sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation, or for determining whether it is wild-type or mutant.
  • nucleotide sequence mutation determination kit examples include those containing the primer pair of the present invention.
  • Preferred are those containing the primer pair M of the present invention and/or the primer pair W of the present invention.
  • Those containing the primer M of the present invention and the primer W of the present invention are preferred.
  • the primer pair of the present invention, the primer pair M of the present invention, and the primer pair W of the present invention according to the kit of the present invention are as described in the section " ⁇ 1. Primer pair of the present invention>", and preferred examples , specific examples, etc. are also the same.
  • the primer pair of the present invention may be in the form of a solution-state test solution such as a suspension suspended in an appropriate buffer, or may be a frozen product or a lyophilized product. good too.
  • concentration of the primer pair of the present invention in the test solution, the solvent of the test solution, and the like may be appropriately set within the range usually used in this field.
  • the kit of the present invention may contain necessary amounts of reagents necessary for carrying out a nucleic acid amplification reaction.
  • reagents necessary for carrying out a nucleic acid amplification reaction for example, in addition to the primer pair of the present invention, nucleoside triphosphate, nucleic acid synthetase, PCR buffer and the like may be further provided.
  • nucleic acid synthetase examples include DNA polymerase, RNA polymerase, reverse transcriptase, and the like.
  • buffers used as the PCR buffer include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, etc. All the buffers used in the case are listed, and the pH thereof is not particularly limited.
  • kit of the present invention may optionally contain substrates (dNTPs, rNTPs, etc.) suitable for the enzyme, such as EvaGreen TM (manufactured by Cosmo Bio Co., Ltd.), SYBR TM Green I (manufactured by Molecular Probe). , ethidium bromide, fluorene, and other double-stranded intercalators, and labeled detection substances such as FAM and TAMRA.
  • substrates dNTPs, rNTPs, etc.
  • substrates dNTPs, rNTPs, etc.
  • EvaGreen TM manufactured by Cosmo Bio Co., Ltd.
  • SYBR TM Green I manufactured by Molecular Probe
  • labeled detection substances such as FAM and TAMRA.
  • stabilizers, preservatives, etc. which do not inhibit the stability of coexisting reagents and the like, and which do not inhibit PCR
  • the kit of the present invention contains an oligonucleotide (wild-type control) having the base sequence of the gene (which may be a partial sequence), including the mutated base sequence of the gene to be detected, and/or the wild-type base of the gene.
  • An oligonucleotide (mutant type control) containing the sequence sequence and the base sequence corresponding to the mutation may be included.
  • Kits of the invention may also include sample amplification curves for determining genotype (wild-type, heterozygous, or homozygous).
  • the kit of the present invention may comprise the necessary amount of the primer pair used in the 1st PCR and the reagents necessary for the 1st PCR.
  • the kit of the present invention includes a nucleic acid amplification reaction procedure using the primers of the present invention, a wild-type control and a mutant control to determine the genotype (wild-type, heterozygous, or homozygous).
  • the above-mentioned "instruction” means the instruction manual, package insert, pamphlet (leaflet), etc. of the above-mentioned kit in which the features, principles, operation procedures, judgment procedures, etc. of the above-mentioned method are substantially described in sentences, diagrams, etc. means
  • kit of the present invention may be equipped with means (for example, cotton swabs, etc.) for collecting test samples.
  • Example 1 Detection of Single Nucleotide Mutation in Human BRAF Gene
  • a single nucleotide substitution (c1799T>A(V600E)) in the human BRAF gene of the present invention was detected by the following method.
  • TIG-3 human fetal normal fibroblasts, JCRB cell bank
  • COLO201 human colon adenocarcinoma cells, ATCC No. CCL-224
  • A375 melanoma cells, ATCC No. CRL-1619
  • Primer pair The following primer pair was designed and synthesized. In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
  • Primer pair without S-merization/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGT-3' (SEQ ID NO: 1) R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCA-3' (SEQ ID NO: 2) ⁇ Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA-3' (SEQ ID NO: 3) R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCT-3' (SEQ ID NO: 4)
  • a single-nucleotide mutation in the human BRAF gene (c1799T>A (V600E)) is a mutation of T to A at position 1799 in the nucleotide sequence of the human BRAF gene.
  • the mutation detection primer pair used in Example 1 is The R primer has the complementary base T of the mutated base A of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
  • the F primer of the pair of primers for detecting mutation type has a complementary base A of the mutated base T of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • EvaGreen TM (manufactured by Biotium): fluorescent reagent for quantitative real-time PCR, manufactured by Cosmo Bio Co., Ltd.
  • KOD FX Neo (manufactured by Toyobo Co., Ltd.): 1 U/ ⁇ L
  • real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in i) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well reaction plate, manufactured by Applied Biosystems Japan Co., Ltd.) and subjected to TaqMan TM PCR thermal cycler detection.
  • Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIGS. 1 to 6 show the results using a primer pair of primers that are not S-conjugated.
  • FIG. 2 shows the results of using a primer pair of primers in which one nucleotide is S-converted.
  • FIG. 3 shows the results of using a primer pair of primers in which 2 bases are S-converted.
  • FIG. 4 shows the results of using a primer pair of primers in which 3 nucleotides are S-converted.
  • FIG. 5 shows the results of using a primer pair of primers in which 4 bases are S-converted.
  • FIG. 6 shows the results of using a primer pair of primers in which 5 nucleotides are S-converted.
  • FIGS. 1 to 6 (1) shows the results of using TIG-3 genomic DNA as a template, (2) shows the results of using COLO201 genomic DNA as a template, and (3) shows the results of A375 genomic DNA. are used as templates.
  • the amplification curve W shows the results of using the wild-type detection primer pair
  • the amplification curve M shows the results of using the mutation-type detection primer pair.
  • TIG-3 cells do not have mutations in the BRAF gene.
  • COLO201 cells have a heterozygous mutation c1799T>A in the BRAF gene.
  • A375 cells have a homozygous c1799T>A mutation in the BRAF gene.
  • the amplification curve using the wild type detection primer pair for the wild type shows the mutation type detection primer pair It rose earlier (earlier detection) than the amplification curve used.
  • the heterozygous type ((2) COLO201)
  • the amplification curve using the wild-type detection primer pair and the amplification curve using the mutant detection primer pair were close to each other.
  • the homozygous type ((3) A375) used the wild type detection primer pair in the amplification curve using the mutant type detection primer pair. It rose ahead of the amplification curve (detected earlier).
  • the heterozygous type and the homozygous type can be clearly distinguished and determined by performing detection using a primer pair of primers in which 2 bases are S-converted or a primer pair of primers in which 3 bases are S-converted. rice field.
  • Example 2 Mutation detection of human ⁇ 1-antitrypsin-1 (PiS) Humans with ⁇ 1-antitrypsin deficiency are known to have the following single base substitutions in the gene. PiS : c.863A>T p.Glu288Val PiZ : c.1096G>A p.Glu366Lys In Example 2, PiS mutation was detected using the primer pair of the present invention.
  • Genomic DNA extraction COLO201 human colon cancer cells, ATCC No. CCL-224
  • A549 human lung cancer cells, ATCC No. CCL-185
  • HepG2 human liver cancer cells, ATCC No. HB-8065
  • a culture solution of MCF7 human breast cancer cells, ATCC No. HTB-22
  • genomic DNA in the collected cells was extracted according to the protocol of the kit.
  • Single base mutation of PiS in ⁇ 1-antitrypsin deficiency is a single nucleotide substitution in which A at position 863 of the nucleotide sequence of ⁇ 1-antitrypsin gene is mutated to T. .
  • the R primer of the primer pair has the complementary base A of the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
  • the F primer of the PiS mutation detection primer pair has the complementary base T of the mutated base A of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • the F primer of the PiS mutation detection primer pair used here has the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the base sequence 5' from the mutated base T in the base sequence of the first strand.
  • the R primer of the PiS mutation detection primer pair has the complementary base A of the mutated base T of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence 3' from the mutated base T of the first strand.
  • the obtained amplification curve is shown in FIG. In FIG. 7, (1) is the result of using COLO201 genomic DNA as a template, (2) is the result of using HepG2 genomic DNA as a template, and (3) is the result of using A549 genomic DNA as a template. and (4) show the results using MCF7 genomic DNA as a template, respectively.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiS mutation type detection primer pair.
  • COLO201 (Fig. 7 (1)) and A549 (Fig. 7 (3)) Amplification curve using a wild-type detection primer pair and the amplification curves using the primer pair for mutation type detection were close to each other. From this, COLO201 and A549 could be determined to have heterozygous PiS mutations.
  • the amplification curve using the wild-type detection primer pair is the amplification using the mutation type detection primer pair. It rose earlier than the curve (detected earlier). From the above, the HepG2 cells and MCF7 cells were determined to be wild-type without PiS mutation.
  • Example 3 Mutation detection of ⁇ 1-antitrypsin-2 (PiZ) In Example 3, PiZ mutation was detected using the primer pair of the present invention.
  • genomic DNA COLO201 human colon cancer cells, ATCC No. CCL-224
  • A549 human lung cancer cells, ATCC No. CCL-185
  • HepG2 human liver cancer cells, ATCC No. HB-8065
  • a culture solution of MCF7 human breast cancer cells, ATCC No. HTB-22
  • genomic DNA in the collected cells was extracted according to the protocol of the kit.
  • Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
  • ⁇ PiZ wild type detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*G-3' (SEQ ID NO: 49)
  • ⁇ PiZ mutation detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*A-3' (SEQ ID NO: 51)
  • R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3' SEQ ID NO: 52
  • the ⁇ 1-antitrypsin deficiency PiZ nucleotide mutation (PiZ: c.1096G>A p.Glu366Lys) is a single nucleotide substitution in which the G at position 1096 in the nucleotide sequence of the ⁇ 1-antitrypsin gene is mutated to A.
  • the R primer of the primer pair has the complementary base T of the mutated base A of the first strand at the 3' end.
  • the rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base A of the first strand.
  • the F primer of the PiZ mutation detection primer pair has the complementary base A of the mutated base T of the second strand at the 3' end.
  • the rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
  • the amplification curve W shows the results of using the wild-type detection primer pair
  • the amplification curve M shows the results of using the mutation-type detection primer pair.
  • Example 4 PiS single nucleotide mutation detection (1) Preparation of sample (control DNA) A partial nucleotide sequence of a region containing the base at position 863 of the human ⁇ 1-antitrypsin gene, and the base corresponding to position 863 is the mutated base T , an oligonucleotide having the base sequence (SEQ ID NO: 54) was designed and synthesized. This oligonucleotide was used as PiS mutant control DNA.
  • an oligonucleotide having a nucleotide sequence (SEQ ID NO: 53), which is a partial nucleotide sequence of a region containing the 863rd nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to the 863rd nucleotide is wild-type A, was designed. and synthesized. This oligonucleotide served as the PiS wild-type control DNA.
  • S-primer pair The same S-primer pair as used in real-time PCR in Example 2 (3) (PiS wild-type detection primer pair: SEQ ID NO: 35-SEQ ID NO: 36 primer pair, PiS mutation type detection A primer pair for SEQ ID NO: 37-SEQ ID NO: 38) was used.
  • PCR reaction solution having the following composition was prepared. Amount of DNA control added: 10 pg for PiS wild-type control DNA only 10 pg for PiS mutant control DNA only 5 pg each for PiS mutant control DNA and PiS wild type control
  • Real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in 1) above is placed in wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad), Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • CFX-96 real-time PCR device
  • FIG. 9(1) shows the results using only PiS wild-type control DNA as a sample. This result is a model for the wild-type PiS gene.
  • FIG. 9(2) shows the results of using a mixed solution of PiS mutant control DNA and PiS wild-type control DNA as a sample. This result is a model for heterozygous PiS mutations.
  • FIG. 9(3) shows the results using only the PiS mutant control DNA as a sample. This result is a model for homozygous PiS mutations.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiS mutation type detection primer pair.
  • the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the ⁇ 1-antitrypsin gene of the subject to be tested in the test sample is wild-type, homozygous PiS mutation, or heterozygous PiS mutation can be discriminated.
  • Example 5 PiZ single nucleotide mutation detection (1) sample preparation A nucleotide sequence ( An oligonucleotide having SEQ ID NO: 56) was designed and obtained by standard methods. This oligonucleotide was used as the PiZ mutant control DNA. In addition, an oligonucleotide having a nucleotide sequence (SEQ ID NO: 55), which is a partial nucleotide sequence of a region containing the 1096th nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to the 1096th nucleotide is a wild-type G, is designed. and obtained by a conventional method. This oligonucleotide served as the PiZ wild-type control DNA.
  • SEQ ID NO: 55 which is a partial nucleotide sequence of a region containing the 1096th nucleotide of the human ⁇ 1-antitrypsin gene and in which the nucleotide corresponding to
  • S-primer pair The same S-primer pair as used in real-time PCR in Example 3 (4) (primer pair for wild type detection: primer pair of SEQ ID NO: 49-SEQ ID NO: 50, for detecting PiZ mutation type Primer pair: primer pair of SEQ ID NO:51-SEQ ID NO:52) was used.
  • PCR reaction solution having the following composition was prepared. Amount of DNA control added: 10 pg for PiZ wild-type control DNA only 10 pg for PiZ mutant control DNA only 5 pg each for PiZ mutant control DNA and PiZ wild type control
  • Real-time PCR 20 ⁇ L of the reaction solution for PCR prepared in 1) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well Reaction Plate, Applied Biosystems Japan), and a thermal cycler for TaqMan TM PCR detection is used.
  • Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIG. 10(1) shows the results using only PiZ wild-type control DNA as a sample. This result is a model for the wild-type PiZ gene.
  • FIG. 10(2) shows the results of using a mixed solution of PiZ mutant control DNA and PiZ wild-type control DNA as a sample. This result is a model for heterozygous PiZ mutations.
  • FIG. 10(3) shows the results of using only the PiZ mutant control DNA as a sample. This result is a model for homozygous PiZ mutations.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PiZ mutation type detection primer pair.
  • the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the ⁇ 1-antitrypsin gene of the test sample is a wild type, a PiZ homozygous mutation, or a PiZ heterozygous mutation can be discriminated.
  • Example 6 Detection of PiS 1 base mutation using human oral swab and saliva sample (1) Preparation of sample 10 ⁇ L of human oral swab (purchased from Busicom Japan) was placed in a tube and treated at 95° C. for 6 minutes in a hot water bath. ⁇ 10 ⁇ L of human saliva (Japanese) was placed in a tube and treated in a hot water bath at 95°C for 6 minutes.
  • PCR amplification 20 ⁇ L of the PCR reaction solution prepared in 2) above was added to a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). It was placed in wells and real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
  • CFX-96 real-time PCR device
  • FIGS. 11 and 12 show the results when mouthwash was used as a sample.
  • FIG. 11 shows the results when mouthwash was used as a sample.
  • FIG. 11 shows the results of mouthwash was used as a sample.
  • FIG. 11 shows the results of saliva was used as a sample.
  • FIG. 12 shows the results when saliva was used as a sample.
  • FIG. 12 shows the results of four specimens, respectively.
  • W indicates the amplification curve obtained using the PiS wild-type detection primer pair
  • M indicates the amplification curve obtained using the PiS mutation type detection primer pair.
  • specimens (1), (2), and (4) were determined to be wild-type (wild-type) with respect to the PiS mutation.
  • the amplification curve W and the amplification curve M rose close to each other (detected at about the same speed), and had the same pattern as in FIG. 9(2). Therefore, specimen (3) was determined to be a heterozygous PiS mutation.
  • specimens (1) to (4) were determined to be wild-type (wild-type) with respect to the PiS mutation.
  • Example 7 PiZ Single Nucleotide Mutation Detection Using Human Oral Wipes and Saliva Samples (1) Preparation of Samples Using the same human oral swabs and human saliva as used in Example 6, pretreatment by heat treatment was performed in the same manner.
  • PCR amplification 20 ⁇ L of the reaction mixture for PCR prepared in 2) above was poured into the wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). , and PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
  • CFX-96 real-time PCR device
  • FIGS. 13 and 14 show the results when a mouthwash was used as a sample.
  • FIG. 13 shows the results when a mouthwash was used as a sample.
  • FIG. 13 shows the results of four specimens, respectively.
  • Fig. 14 shows the results when saliva was used as a sample.
  • (1) to (4) show the results of four specimens, respectively.
  • W indicates the amplification curve obtained using the PiZ wild-type detection primer pair
  • M indicates the amplification curve obtained using the PiZ mutation type detection primer pair.
  • specimens (1) to (4) were all determined to be wild-type for the PiZ mutation.
  • specimens (1) to (4) were all determined to be wild-type for the PiZ mutation.
  • Example 8 Detection of Nucleotide Sequence Mutations in Cats In cats, the following single-nucleotide substitutions in genes are known.
  • PKD1 c.9864C>A
  • PKLR c.693 + 304G > A
  • the primer pairs of the present invention were used to detect feline PKD1 and PKLR gene mutations.
  • Example 2 (2) Preparation of PCR reaction mixture and 1st-PCR
  • the sample-derived genomic DNA was used as a template to prepare a PCR reaction solution and perform PCR amplification to obtain a PCR amplification product.
  • a region containing base 9864 of the PKD1 gene is amplified.
  • a region containing bases at positions 693+304 of the PKLR gene is amplified.
  • ⁇ PKD1 wild type detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*C-3' (SEQ ID NO: 61) R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*G-3' (SEQ ID NO: 62) ⁇ PKD1 mutation detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*A-3' (SEQ ID NO: 63) R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*T-3' (SEQ ID NO: 64) ⁇ PKLR wild type detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*G-3' (SEQ ID NO: 65) R primer: 5'-GTCAGGGGCGAGCCGGGGGCAGA*G*T*C-3' (SEQ ID NO: 66) ⁇ PKLR mutation detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*A
  • FIGS. 15 and 16 Results The obtained amplification curves are shown in FIGS. 15 and 16.
  • FIG. 15 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene and PKLR gene have been confirmed to be wild-type.
  • Fig. 16 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene was confirmed to be wild-type and the PKLR gene was confirmed to be mutant.
  • the amplification curve W shows the results using the wild-type detection primer pair
  • the amplification curve M shows the results using the PKD1 mutation detection primer pair or the PKLR mutation detection primer pair.
  • a primer pair for detecting the PKD1 wild-type is used.
  • the amplification curve using the PKD1 mutation type rose earlier than the amplification curve using the primer pair for detecting the PKD1 mutation (detected earlier).
  • the amplification curve using the PKLR wild-type detection primer pair rose earlier than the amplification curve using the PKLR mutation type detection primer pair (earlier detection was done). Based on the above, it was determined that the subject cat was a wild-type cat with no PKD1 mutation or PKLR mutation.
  • the PKD1 gene is wild-type, and when genomic DNA derived from a cat that has been confirmed to have a PKLR gene mutation is used as a template (Fig. 16), as is clear from Fig. 16 (1), a PKD1 wild-type detection primer The amplification curve using the pair rose earlier (detected earlier) than the amplification curve using the primer pair for detecting the PKD1 mutation type. On the other hand, as is clear from FIG. 15(2), the amplification curve using the PKLR wild-type detection primer pair and the amplification curve using the PKLR mutation type detection primer pair were close to each other. From the above, it was determined that the subject cat was wild-type with respect to PKD1, but had a PKLR mutation, and that the mutation was heterozygous.
  • Example 9 Detection of Nucleotide Sequence Mutations in Dogs Dogs are known to have a 4-nucleotide deletion mutation that lacks GTTT at positions 4411950-4411953 of the VPS13B gene (g.4411950_4411953delGTTT).
  • the primer pair of the present invention was used to detect canine VPS13B gene mutation.
  • Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
  • ⁇ VPS13B wild type detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T-3' (SEQ ID NO: 71) R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3' (SEQ ID NO: 72) ⁇ VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73) R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3' (SEQ ID NO: 74)
  • amplification curve W shows the results using the wild-type detection primer pair
  • amplification curve M shows the results using the VPS13B mutation detection primer pair.
  • the amplification curve W and the amplification curve M using samples derived from Chihuahua and seeds are overlapped, so they appear to be one.
  • Example 10 Determination of N501Y and E484K Mutations of SARS-CoV-2 Virus We focused on the following mutations of SARS-CoV-2 virus.
  • N501Y mutation Mutation identified in British, South African, and Brazilian variants. It is a single nucleotide substitution of A23063T in the nucleotide sequence of the SARS-CoV-2 virus (GenBank, Accession No. MN908947.3). That is, it is a mutation in which the 23063rd base of the RNA of the SARS-CoV-2 virus is mutated from A to T, and a mutation in which the 501st amino acid asparagine of the spike protein is mutated to tyrosine.
  • the N501Y mutant of the SARS-CoV-2 virus causes protein mutations in the receptor-binding portion of the spike protein, resulting in increased binding affinity with the human and mouse receptor ACE2.
  • E484K mutation A mutation identified in the South African variant and the Brazilian variant. It is a single nucleotide substitution of G23012A in the nucleotide sequence of the SARS-CoV-2 virus. That is, the 23012th base of the SARS-CoV-2 virus RNA is mutated from G to A, and the 484th amino acid glutamic acid of the spike protein is mutated to lysine.
  • the E484K variant of the SARS-CoV-2 virus may neutralize antibodies generated by vaccination.
  • F primer for E484K mutation 5'-CCAGATGATTTTACAGGCTGCGTTATAGC-3' (SEQ ID NO: 77)
  • R primer 5'-CAAACAGTTGCTGGTGCATGTAGAAGTTC-3' (SEQ ID NO: 78)
  • This 1st-PCR amplifies the region containing the mutated portion of each mutation in the nucleic acid of the SARS-CoV-2 viral gene.
  • the region containing the portion corresponding to each mutation portion is amplified.
  • N501Y mutation detection/N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80) ⁇ N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81) R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
  • E484K mutation detection/E484K wild type detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*G-3' (SEQ ID NO: 83) R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*C-3' (SEQ ID NO: 84) ⁇ E484K mutation detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*A-3' (SEQ ID NO: 85) R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*T-3' (SEQ ID NO: 86)
  • ii) real-time PCR That is, 20 ⁇ L of the PCR reaction solution prepared in i) above was placed in the wells of a 96-well reaction plate (Hard-Shell TM Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad).
  • Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubating at 94°C for 2 minutes, reaction at 98°C for 5 seconds and at 68°C for 10 seconds was repeated for 40 cycles. Then, fluorescence intensity derived from EvaGreen TM was measured.
  • FIG. 18 shows the results of detecting the N501Y mutation.
  • FIG. 19 shows the results of detecting the E484K mutation.
  • FIGS. 18 and 19 (1) the results obtained using DNA having the base sequence of the wild strain of the SARS-CoV-2 virus, (2) the N501Y mutation of the SARS-CoV-2 virus or Results obtained using DNA having the base sequence of the E484K mutation are shown respectively.
  • W indicates the results obtained using the wild-type detection primer pair
  • M indicates the results obtained using the mutation-type detection primer pair.

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Abstract

The present invention relates to: "a primer pair comprising a forward primer in which a phosphodiester bond on the 3' side of 1-4 contiguous nucleotides from the second base from the 3' end toward the 5' side is phosphorothioated (S-ized), and a reverse primer in which a phosphodiester bond on the 3' side of 1-4 contiguous nucleotides from the second base from the 3' end toward the 5'side is S-ized; and a method for determining variation in a base sequence using the primer pair."

Description

[規則26に基づく補充 09.05.2022]　プライマー対、塩基配列変異の判定方法及び塩基配列の変異判定用キット[Replenishment based on Rule 26 09.05.2022] Primer pair, base sequence mutation determination method, and base sequence mutation determination kit
　本発明は、プライマー対、塩基配列の変異の判定方法及び塩基配列の変異判定用キットに関する。 The present invention relates to a primer pair, a method for determining nucleotide sequence mutation, and a kit for determining nucleotide sequence mutation.
　病気の罹り易さ、薬の効き易さ、副作用の出易さ等が個体間で異なる原因の一つに、遺伝子の変異（塩基配列の変異）が関与している場合がある。そこでこの遺伝子配列の違いに着目し、体質にあった治療法を探る研究が進められている。また、遺伝子変異は、多数の疾患の遺伝マーカーともなり得る。
　すなわち、遺伝子変異の解明は臨床的に重要であり、そのため遺伝子変異を検出できる分析法の確立が望まれている。 Gene mutation (base sequence mutation) may be involved in one of the causes of individual differences in disease susceptibility, drug efficacy, side effect susceptibility, and the like. Therefore, research is underway to search for a treatment method suitable for the constitution, focusing on the difference in this gene arrangement. Gene mutations can also serve as genetic markers for many diseases.
That is, elucidation of gene mutation is clinically important, and therefore establishment of an analytical method capable of detecting gene mutation is desired.
　しかしながら、変異遺伝子の存在を検出し、判定するためには、膨大なゲノム塩基配列中のわずか数塩基の違いを検出する必要があることから、非常に高い特異性が要求される。 However, in order to detect and determine the existence of mutated genes, it is necessary to detect differences in just a few bases in the huge genome base sequence, so extremely high specificity is required.
　従来の一般的な核酸配列分析技術としては、例えば核酸配列決定法、RFLP（制限酵素切断長多型）法、ASP（アレル特異的プライマー）法、ASO（アレル特異的オリゴプローブ）法、１塩基伸長法等が知られている。 Conventional general nucleic acid sequence analysis techniques include, for example, nucleic acid sequencing, RFLP (restriction enzyme cleavage polymorphism), ASP (allele-specific primer), ASO (allele-specific oligoprobe), single base A decompression method and the like are known.
　これらの方法は、予め変異や多型を含む領域をポリメラーゼ連鎖反応（Polymerase Chain Reaction、PCR）等の核酸増幅技術を利用して増幅しておいてから検出する手法である。そのため、まず特異的な増幅産物を得ることが遺伝子変異を特異的に検出するために非常に重要である。 These methods are methods in which a region containing mutations or polymorphisms is amplified in advance using a nucleic acid amplification technology such as the polymerase chain reaction (PCR) and then detected. Therefore, it is very important to first obtain a specific amplification product in order to specifically detect genetic mutations.
　また、ヌクレオチドのホスホジエステル結合の一部又は全てがホスホロチオエート化されたフォワードプライマーを用いたPCRを行って遺伝子変異を検出する方法も知られている（特許文献１）。 Also known is a method of detecting gene mutation by performing PCR using a forward primer in which some or all of the phosphodiester bonds of nucleotides are phosphorothioated (Patent Document 1).
　特許文献１には、「プライマーから伸長するヌクレオチド鎖が実質的にエキソヌクレアーゼ活性に対して耐性であるように、一つ以上の化学修飾されたヌクレオチド、塩基又はホスホジエステル結合を含むプライマー」と、「固体支持体に固定化されたセンス配列及びそれに相補的配列を有するヌクレオチド配列を含むプライマー」を用いて、標的ヌクレオチド配列を増幅し増幅産物を生成する工程を含み、その結果に基づいて生物の標的ヌクレオチドがホモ接合であるかヘテロ接合であるかを検出し、判定する方法が開示されている。 In Patent Document 1, ``a primer containing one or more chemically modified nucleotides, bases, or phosphodiester bonds so that the nucleotide chain extending from the primer is substantially resistant to exonuclease activity,'' Using "a primer comprising a nucleotide sequence having a sense sequence immobilized on a solid support and a sequence complementary thereto", a step of amplifying a target nucleotide sequence to generate an amplification product, based on the result Methods are disclosed for detecting and determining whether a target nucleotide is homozygous or heterozygous.
特開２０１０－０２２３８３号公報JP 2010-022383 A
　しかし、上記した如き従来の核酸増幅反応による方法では、プライマーにミスマッチが存在する場合でも伸長反応が起きてしまう場合があり、変異遺伝子検出及び判定の正確性に問題があった。 However, in the above-described conventional method based on nucleic acid amplification reaction, an extension reaction may occur even when there is a mismatch in the primer, and there is a problem with the accuracy of mutant gene detection and determination.
　本発明は、上記したごとき状況に鑑みなされたもので、正確な遺伝子変異の判定に用いられるプライマー対、正確な遺伝子変異の判定方法、及び、正確な遺伝子変異の判定に用いられる塩基配列の変異判定用キットの提供を課題とする。 The present invention has been made in view of the above circumstances, and provides a primer pair used for accurate determination of genetic mutation, an accurate method for determining genetic mutation, and a nucleotide sequence mutation used for accurate determination of genetic mutation. The object is to provide a determination kit.
　本発明者は、鋭意研究の結果、フォワードプライマーとリバースプライマーの両方が、３'末端から２番目の塩基から上流側の連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がホスホロチオエート化されたものであるプライマー対を用いた核酸増幅反応を行ったところ、正しい塩基配列の検出及び判定が可能となることを見出し、本発明を完成するに到った。 As a result of intensive research, the present inventors have found that both the forward primer and the reverse primer have phosphorothioated phosphodiester bonds on the 3' side of 1 to 4 consecutive nucleotides upstream from the second base from the 3' end. When a nucleic acid amplification reaction was carried out using the primer pair thus obtained, the inventors found that the correct base sequence can be detected and determined, and have completed the present invention.
　すなわち、本発明は、代表的には以下の発明を含んでなる。
［１］
３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がホスホロチオエート化（Ｓ化）されているフォワードプライマーと、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーとのプライマー対。
［２］
　プライマー対Ｍ：
変異を有する二本鎖核酸の一方の鎖（第２鎖）にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第２鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
上記変異を有する二本鎖核酸のもう一方の鎖（第１鎖）にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第１鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対、
　プライマー対Ｗ：
野生型の二本鎖核酸の一方の鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
野生型の二本鎖核酸のもう一方の鎖（野生型の第１鎖）にアニールするプライマーであり、上記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。
［３］
上記プライマー対Ｍに係る塩基配列の変異が塩基の置換又は挿入であり、上記プライマー対Ｍが下記のものである、上記[２]に記載のプライマー対：
　上記変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第２鎖の変異配列の相補配列を３’末端側に有し、その他の塩基配列は上記第２鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
　上記変異を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第１鎖の変異配列の相補配列を３’末端側に有し、その他の塩基配列は上記第１鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。
［４］
上記プライマー対Ｍに係る塩基配列の変異が塩基の欠損であり、上記プライマー対Ｍが下記のものである、上記[２]に記載のプライマー対：
　上記プライマー対Ｍに係る塩基配列の変異が塩基の欠損であり、上記プライマー対Ｍが下記のものである、請求項２に記載のプライマー対：
　上記欠損を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記欠損を含む領域にアニールし、上記第２鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端に有し、その他の塩基配列は上記第２鎖の上記欠損に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
　上記欠損を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記欠損を含む領域にアニールし、上記第１鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端に有し、その他の塩基配列は上記第１鎖の上記欠損に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。
［５］
上記プライマー対Ｗが下記のものである、上記[２]～[４]の何れか一つに記載のプライマー対：
　上記プライマー対Ｗが下記のものである、請求項２に記載のプライマー対：
　野生型の二本鎖核酸の一方の鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
　上記野生型の二本鎖核酸のもう一方の鎖（野生型の第１鎖）にアニールするプライマーであり、上記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。
［６］
プライマーの３'末端から２番目の塩基から５’側に向けての連続する３個のヌクレオチドの３'側のホスホジエステル結合がＳ化されている、上記[１]～[５]の何れか一つに記載のプライマー対。
［７］
上記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、上記[１]～[３]、及び[６]の何れか一つに記載のプライマー対。
［８］
被検試料の核酸を鋳型として用い、上記[１]～[７]の何れか一つに記載のプライマー対を用いた核酸増幅反応を行って反応産物を検出し、得られた検出結果をもとに、上記核酸の塩基配列の変異を判定する、塩基配列の変異の判定方法。
［９］
上記[２]～[７]の何れか一つに記載のプライマー対を用い、核酸増幅反応を行って反応産物を検出し得られた検出結果をもとに上記核酸の塩基配列の変異を判定する方法が下記の工程を含む、上記[８]に記載の判定方法：
　（１）被検試料の核酸を鋳型として用い、上記[２]～[４]、[６]～[７]の何れか一つに記載のプライマー対Ｍを用いた核酸増幅反応を行い、反応産物を検出する工程、
　（２）上記(１)の工程で用いたものと同じ被検試料の核酸を鋳型として用い、上記[２]、[５]～[７]の何れか一つに記載のプライマー対Ｗを用いた核酸増幅反応を行い、反応産物を検出する工程、
　（３）上記(１)及び(２)の工程で得られた検出結果をもとに、被検試料の核酸の塩基配列の変異を判定する工程。
［１０］
被検試料の核酸を鋳型として用いた核酸増幅反応を行い、塩基配列の変異が存在し得る領域を増幅し、得られた増幅産物を鋳型として用いる、上記[８]又は[９]に記載の判定方法。
［１１］
上記塩基配列の変異の判定が、被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かを判定すること、又は野生型か変異型かを判定することである、上記[８]～[１０]の何れか一つに記載の判定方法。
［１２］
上記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、上記[８]～[１１]の何れか一つに記載の判定方法。
［１３］
上記[１]～[７]の何れか一つに記載のプライマー対を含む、塩基配列の変異判定用キット。
［１４］
被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かの判定用、又は野生型か変異型かの判定用である、上記[１３]に記載のキット。
［１５］
上記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、上記[１３]又は[１４]に記載のキット。 Specifically, the present invention typically includes the following inventions.
[1]
A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is phosphorothioated (S), and a forward primer from the 3' end A primer pair with a reverse primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base toward the 5' side is S-converted.
[2]
Primer pair M:
A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side. A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-formed,
A primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of a reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed,
Primer pair W:
A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-type second strand, and placing a complementary sequence of the base sequence in the wild-type second strand corresponding to the mutation in the mutant second strand on the 3′ end side A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed,
A primer that anneals to the other strand of the wild-type double-stranded nucleic acid (wild-type first strand), and for mutation of the first strand of the mutant double-stranded nucleic acid (mutant-type first strand) Annealing to the region containing the base sequence in the corresponding wild-type first strand, and 3′ the complementary sequence of the base sequence in the wild-type first strand corresponding to the mutation in the mutant first strand A reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side, which has a terminal side and is S-ified. primer pair.
[3]
The primer pair according to [2] above, wherein the mutation in the base sequence of the primer pair M is a base substitution or insertion, and the primer pair M is as follows:
A primer that anneals to the second strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the second strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, and has 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side. a forward primer in which the phosphodiester bond on the 3′ side of the nucleotide is S-converted;
A primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side. A primer pair of reverse primers in which the phosphodiester bond on the 3' side of the nucleotide is S-converted.
[4]
The primer pair according to [2] above, wherein the mutation in the base sequence of the primer pair M is a base deletion, and the primer pair M is as follows:
The primer pair according to claim 2, wherein the mutation in the nucleotide sequence of the primer pair M is a deletion of a base, and the primer pair M is:
A primer that anneals to the second strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5'-side nucleotide sequence adjacent to the deletion of the second strand to the 3' end. and the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, and extends from the second base from the 3' end to the 5' side a forward primer in which the phosphodiester bond on the 3′ side of consecutive 1 to 4 nucleotides is S-formed;
A primer that anneals to the first strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5'-side nucleotide sequence adjacent to the deletion of the first strand to the 3' end. and the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the first strand, and from the second base from the 3' end to the 5' side A pair of reverse primers in which the phosphodiester bond on the 3' side of consecutive 1 to 4 nucleotides is S-converted.
[5]
The primer pair according to any one of [2] to [4] above, wherein the primer pair W is:
3. The primer pair of claim 2, wherein said primer pair W is:
A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to the region containing the base sequence in the wild-type second strand, and placing a complementary sequence of the base sequence of the wild-type second strand corresponding to the mutation of the mutant second strand on the 3' end side and the other nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the nucleotide sequence of the wild-type second strand corresponding to the mutation of the mutant second strand, a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 'end toward the 5' side is S-formed;
A primer that anneals to the other strand (wild-type first strand) of the wild-type double-stranded nucleic acid, and mutation of the first strand (mutant-type first strand) of the mutant double-stranded nucleic acid Annealing to the region containing the base sequence in the wild-type first strand corresponding to the 3' complementary sequence of the base sequence of the wild-type first strand corresponding to the mutation in the mutant first strand The rest of the nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation of the mutant first strand. A pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted.
[6]
Any of the above [1] to [5], wherein the phosphodiester bond on the 3' side of three consecutive nucleotides from the 2nd base from the 3' end of the primer toward the 5' side is S-ified. Primer pairs according to one.
[7]
The primer pair according to any one of [1] to [3] and [6] above, wherein the mutation is PiS mutation or PiZ mutation in the α1-antitrypsin gene.
[8]
Using the nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair according to any one of [1] to [7] above to detect the reaction product, and the resulting detection results are also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid.
[9]
A nucleic acid amplification reaction is performed using the primer pair according to any one of the above [2] to [7], and the reaction product is detected. Based on the detection results obtained, the mutation in the base sequence of the nucleic acid is determined. The determination method according to [8] above, wherein the method includes the following steps:
(1) Using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M according to any one of [2] to [4] and [6] to [7] above, detecting the product;
(2) Using the nucleic acid of the same test sample as that used in the step (1) above as a template, and using the primer pair W according to any one of [2], [5] to [7] above. a step of performing a nucleic acid amplification reaction and detecting a reaction product;
(3) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (1) and (2).
[10]
The above-mentioned [8] or [9], wherein a nucleic acid amplification reaction is performed using the nucleic acid of the test sample as a template, a region in which a nucleotide sequence mutation may exist is amplified, and the resulting amplification product is used as a template. judgment method.
[11]
Determination of the mutation in the base sequence is to determine whether the base sequence of the nucleic acid in the test sample is a wild type, homozygous mutation, or heterozygous mutation, or to determine whether it is a wild type or a mutant type. , the determination method according to any one of the above [8] to [10].
[12]
The determination method according to any one of [8] to [11] above, wherein the mutation is PiS mutation or PiZ mutation in the α1-antitrypsin gene.
[13]
A nucleotide sequence mutation determination kit comprising the primer pair according to any one of [1] to [7] above.
[14]
The kit according to [13] above, which is for determining whether the base sequence of the nucleic acid of the test sample is wild-type, homozygous mutation, or heterozygous mutation, or for determining whether it is wild-type or mutant.
[15]
The kit according to [13] or [14] above, wherein the mutation is a PiS mutation or a PiZ mutation in the α1-antitrypsin gene.
　本発明のプライマー対を用いた塩基配列の変異の判定方法によれば、検出対象の遺伝子の塩基配列変異を正確に検出できる。更に、ヘテロ接合型変異又はホモ接合型変異の判定を行える。
　本発明の塩基配列の変異の判定方法によれば、検出対象の遺伝子の塩基配列変異を正確に検出できる。更に、ヘテロ接合型変異又はホモ接合型変異の判定を行える。
　本発明のキットを用いた塩基配列の変異の判定方法によれば、検出対象の遺伝子の塩基配列変異を正確に検出できる。更に、ヘテロ接合型変異又はホモ接合型変異の判定を行える。 According to the method for determining nucleotide sequence mutations using the primer pair of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made.
According to the method for determining nucleotide sequence mutations of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made.
According to the method for determining nucleotide sequence mutations using the kit of the present invention, nucleotide sequence mutations in genes to be detected can be accurately detected. Additionally, determination of heterozygous or homozygous mutations can be made.
実施例１で得られた、Ｓ化していないプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of non-S-conjugated primers obtained in Example 1. FIG. 実施例１で得られた、１塩基がＳ化されたプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers obtained in Example 1, in which one nucleotide is S-converted. 実施例１で得られた、２塩基がＳ化されたプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which two bases are S-modified obtained in Example 1. FIG. 実施例１で得られた、３塩基がＳ化されたプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 3 bases are S-modified obtained in Example 1. FIG. 実施例１で得られた、４塩基がＳ化されたプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers in which 4 bases are S-modified obtained in Example 1. FIG. 実施例１で得られた、５塩基がＳ化されたプライマーのプライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。1 is an amplification curve obtained by performing real-time PCR using a primer pair of primers having 5 nucleotides in S, obtained in Example 1. FIG. 実施例２で得られた、COLO201、A549、HepG2及びMCF7のゲノムDNAのPiS変異を検出対象とし、本発明のPiS変異を検出するＳ化プライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, targeting the PiS mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 2. curve. 実施例３で得られた、COLO201、A549、HepG2及びMCF7のゲノムDNAのPiZ変異を検出対象とし、本発明のPiZ変異を検出するＳ化プライマー対を用いたリアルタイムPCRを行って得られた増幅曲線である。Amplification obtained by performing real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, targeting the PiZ mutation in the genomic DNA of COLO201, A549, HepG2, and MCF7 obtained in Example 3. curve. 実施例４で得られた、PiS変異の野生型、ホモ接合型、又はヘテロ接合型の増幅曲線のモデルである。4 is a model of amplification curves of wild-type, homozygous, or heterozygous PiS mutations obtained in Example 4. FIG. 実施例５で得られた、PiZ変異の野生型、ホモ接合型、又はヘテロ接合型の増幅曲線のモデルである。6 is a model of amplification curves for wild-type, homozygous, or heterozygous PiZ mutations obtained in Example 5. FIG. 実施例６で得られた、口腔拭い液由来のゲノムDNAのPiS変異を検出対象とし、本発明のPiS変異を検出するＳ化プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in the genomic DNA derived from the oral swab obtained in Example 6 as the detection target. 実施例６で得られた、唾液由来のゲノムDNAのPiS変異を検出対象とし、本発明のPiS変異を検出するＳ化プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiS mutation of the present invention, with the PiS mutation in saliva-derived genomic DNA obtained in Example 6 as the detection target. 実施例７で得られた、口腔拭い液を由来のゲノムDNAのPiZ変異を検出対象とし、本発明のPiZ変異を検出するＳ化プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in the genomic DNA derived from the oral swab obtained in Example 7 as the detection target. 実施例７で得られた、唾液由来のゲノムDNAのPiZ変異を検出対象とし、本発明のPiZ変異を検出するＳ化プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 2 shows an amplification curve obtained by real-time PCR using the S-primer pair for detecting the PiZ mutation of the present invention, with the PiZ mutation in saliva-derived genomic DNA obtained in Example 7 as a detection target. 実施例８で得られた、PKD1遺伝子及びPKLR遺伝子が野生型であることが確認されているPKD1遺伝子及びPKLR遺伝子が野生型であることが確認されているネコ由来のゲノムDNAのPKD1遺伝子及びPKLR遺伝子の変異を検出対象とし、本発明のPKD1検出用プライマー対又はPKLR検出用プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。The PKD1 gene and PKLR of genomic DNA derived from a cat in which the PKD1 gene and PKLR gene have been confirmed to be wild-type, obtained in Example 8 Fig. 2 shows an amplification curve obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting gene mutations. 実施例８で得られた、PKD1遺伝子が野生型でPKLR遺伝子が変異型であることが確認されているPKD1遺伝子が野生型で、PKLR遺伝子が変異型であることが確認されているネコ由来のゲノムDNAのPKD1遺伝子及びPKLR遺伝子の変異を検出対象とし、本発明のPKD1検出用プライマー対又はPKLR検出用プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。The PKD1 gene obtained in Example 8 was confirmed to be wild-type and the PKLR gene was of a mutant type. Fig. 2 shows amplification curves obtained by real-time PCR using the PKD1 detection primer pair or the PKLR detection primer pair of the present invention, targeting mutations in the PKD1 gene and the PKLR gene in genomic DNA. 実施例９で得られた、イヌ由来のゲノムDNAのVPS13B変異を検出対象とし、本発明のVPS13B検出用プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 10 is an amplification curve obtained by real-time PCR using the primer pair for VPS13B detection of the present invention, with the VPS13B mutation in canine genomic DNA obtained in Example 9 as the detection target. 実施例１０で得られた、SARS-CoV-2ウイルス由来ゲノムRNAのSN501Y変異を検出対象とし、本発明のSN501Y変異検出用プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。Fig. 10 shows an amplification curve obtained by real-time PCR using the SN501Y mutation detection primer pair of the present invention, with the SN501Y mutation of the SARS-CoV-2 virus-derived genomic RNA obtained in Example 10 as the detection target. 実施例１０で得られた、SARS-CoV-2ウイルス由来ゲノムRNAのびE484K変異を検出対象とし、本発明のE484K変異検出用プライマー対を用いたリアルタイムPCRで得られた増幅曲線である。10 shows an amplification curve obtained by real-time PCR using the E484K mutation detection primer pair of the present invention, with the SARS-CoV-2 virus-derived genomic RNA and the E484K mutation as detection targets, obtained in Example 10.
　本発明において、塩基配列の変異を検出する対象の遺伝子に関し、塩基配列の変異が起きていない塩基配列を「野生型の塩基配列」という。
　本発明において「変異」とは、塩基配列の変異を検出する対象の遺伝子の、野生型とは異なる塩基配列、又は上記塩基配列を有することをいう。「変異」の例としては、塩基配列の変異を検出する対象の遺伝子の野生型の塩基配列に対して、塩基の置換、欠失、挿入等が挙げられる。変異した塩基の数は特に限定されない。一ヶ所の変異当り１～４個の塩基の変異、好ましくは１～３個の変異、より好ましくは１個の変異が挙げられる。 In the present invention, a nucleotide sequence in which no nucleotide sequence mutation occurs is referred to as a "wild-type nucleotide sequence" with respect to the target gene whose nucleotide sequence mutation is to be detected.
In the present invention, the term “mutation” refers to a base sequence different from that of the wild type of the target gene whose base sequence mutation is to be detected, or having the above-mentioned base sequence. Examples of "mutation" include base substitution, deletion, insertion, and the like with respect to the wild-type base sequence of the gene whose base sequence mutation is to be detected. The number of mutated bases is not particularly limited. 1 to 4 base mutations, preferably 1 to 3 base mutations, more preferably 1 mutation are included per mutation.
　本発明において検出対象の遺伝子の塩基配列が変異を持たない場合、その塩基配列は野生型であるという。一方、検出対象の遺伝子の塩基配列が変異を持っている場合、その塩基配列は変異型である、という。 In the present invention, when the base sequence of the gene to be detected does not have mutations, the base sequence is said to be wild-type. On the other hand, when the nucleotide sequence of the gene to be detected has mutations, the nucleotide sequence is said to be of a mutant type.
　また、本発明において遺伝子型を判定する場合、対立遺伝子の両方の塩基配列が野生型である場合を野生型と言う。対立遺伝子の片方の塩基配列が変異を含む場合をヘテロ接合型と言う。また、対立遺伝子の両方の塩基配列が同じ変異を含む場合をホモ接合型と言う。本発明における「変異」とは、このヘテロ接合型及びホモ接合型の遺伝子型を含む。 In addition, when genotypes are determined in the present invention, a case where both base sequences of alleles are wild type is referred to as wild type. Heterozygosity is when one of the alleles contains the mutation. Also, when both nucleotide sequences of alleles contain the same mutation, it is called homozygous. "Mutation" in the present invention includes this heterozygous and homozygous genotype.
　本発明に係る塩基配列の変異を検出し、判定する対象の遺伝子（変異の形態）としては、具体的には、例えばヒトの場合、BRAF(B-Raf proto-oncogene, serine/threonine kinase)（c1799T＞A(V600E)），α1－アンチトリプシン［Pis（c.863A>T p.Glu288Val）、PiZ（c.1096G>A p.Glu366Lys）］等、ネコの場合、PKD1（c. 9864C>A），PKLR（c. 693 + 304G>A）等、イヌの場合、VPS13B（g.4411950_4411953delGTTT），MFSD8，HEXB，GLB1等、ウイルス，例えばSARS-CoV-2の場合、N501Y（A23063T），E484K（G23012A）等が挙げられる。 Specific examples of target genes (mutation forms) for detecting and determining nucleotide sequence mutations according to the present invention include, for example, BRAF (B-Raf proto-oncogene, serine/threonine kinase) ( c1799T>A (V600E)), α1-antitrypsin [Pis (c.863A>T p.Glu288Val), PiZ (c.1096G>A p.Glu366Lys)], etc. In cats, PKD1 (c. 9864C>A ), PKLR (c. 693 + 304G>A), etc. For dogs, VPS13B (g.4411950_4411953delGTTT), MFSD8, HEXB, GLB1, etc., viruses, for example, SARS-CoV-2, N501Y (A23063T), E484K ( G23012A) and the like.
　本発明に係る塩基配列の変異を検出し、判定する対象の遺伝子において、ホモ接合型変異又はヘテロ接合型変異等の遺伝子型の変異を判定する対象の遺伝子としては、例えばBRAF、Pis、PiZ等が挙げられる。 In the target genes for detecting and determining nucleotide sequence mutations according to the present invention, target genes for determining genotype mutations such as homozygous mutations or heterozygous mutations include, for example, BRAF, Pis, PiZ, etc. are mentioned.
　本発明に係る「相補塩基」、及び「相補配列」とは、通常この分野で用いられている意味で用いられる。即ち、「相補塩基」とは、ある塩基に対して相補的な塩基をいう。また、「相補鎖」とは、ある核酸鎖に対して相補的な塩基配列を有する核酸鎖を言う。「相補配列」とは、ある塩基配列に対して相補的な塩基配列を言う。 The terms "complementary base" and "complementary sequence" according to the present invention are used in the meaning usually used in this field. That is, "complementary base" refers to a base that is complementary to a certain base. A "complementary strand" refers to a nucleic acid strand having a base sequence complementary to a given nucleic acid strand. A "complementary sequence" refers to a base sequence that is complementary to a given base sequence.
　尚、特に断りのない限り、本明細書において「塩基配列」といった場合には、１塩基の場合と複数の塩基の塩基配列の両方の意味を含む。 Unless otherwise specified, the term "nucleotide sequence" as used herein includes both a single base sequence and a multiple base sequence.
　本明細書で用いられるその他の語句について、下図[Ｓ１]～［Ｓ３］をもとに説明する。 Other terms used in this specification will be explained based on the diagrams [S1] to [S3] below.
[図Ｓ１] 変異が置換の場合
Figure JPOXMLDOC01-appb-I000001
[Figure S1] When the mutation is a substitution
Figure JPOXMLDOC01-appb-I000001
　図Ｓ１は、ある遺伝子に関し、野生型の二本鎖核酸の一方の鎖と、変異型の二本鎖核酸の一方の鎖であって、塩基配列Ｘが塩基配列Ｙに置換している変異を有する鎖を図示したものである。
　図Ｓ１において「変異配列」とは、変異を有する核酸のＹの塩基配列を表す。
　「変異に対応する塩基配列」とは、野生型の核酸のＸの塩基配列を表す。
　「変異を含む領域」とは、変異を有する核酸の、Ｙを含む塩基配列の領域（＊）を表す。
　「変異に対応する(野生型の)塩基配列を含む領域」とは、野生型の核酸の、Ｘを含む塩基配列の領域（＋）を表す。 FIG. S1 shows a mutation in which one strand of a wild-type double-stranded nucleic acid and one strand of a mutant double-stranded nucleic acid are substituted for a base sequence X with a base sequence Y for a gene. Figure 10 illustrates a chain with
In FIG. S1, "mutant sequence" represents the base sequence of Y of the nucleic acid with mutation.
The “nucleotide sequence corresponding to the mutation” refers to the X nucleotide sequence of the wild-type nucleic acid.
The term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
A “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing X in a wild-type nucleic acid.
[図Ｓ２] 変異が挿入の場合
 
Figure JPOXMLDOC01-appb-I000002
[Figure S2] When the mutation is an insertion
Figure JPOXMLDOC01-appb-I000002
　図Ｓ２は、ある遺伝子に関し、野生型の二本鎖核酸の一方の鎖と、変異型の二本鎖核酸の一方の鎖であって、塩基配列Ｘ１と塩基配列Ｘ２の間に塩基配列Ｙが挿入された変異を有する鎖を図示したものである。
　図Ｓ２において「変異配列」とは、変異を有する核酸のＹの塩基配列を表す。
　「変異に対応する塩基配列」とは、野生型の核酸のＸ１及び／又はＸ２の塩基配列を表す。
　「変異を含む領域」とは、変異を有する核酸の、Ｙを含む塩基配列の領域（＊）を表す。
　「変異に対応する（野生型の）塩基配列を含む領域」とは、野生型の核酸の、Ｘ１及びＸ２を含む塩基配列の領域（＋）を表す。 FIG. S2 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the base sequence Y is between the base sequence X1 and the base sequence X2. A diagram of the strand with the mutations inserted.
In FIG. S2, "mutant sequence" represents the base sequence of Y of the nucleic acid with mutation.
The “nucleotide sequence corresponding to the mutation” represents the nucleotide sequence of X1 and/or X2 of the wild-type nucleic acid.
The term “mutation-containing region” refers to the region (*) of the nucleotide sequence containing Y in the nucleic acid having the mutation.
“A region containing a (wild-type) base sequence corresponding to a mutation” refers to a base sequence region (+) containing X1 and X2 of a wild-type nucleic acid.
[図Ｓ３] 変異が欠損の場合
Figure JPOXMLDOC01-appb-I000003
[Figure S3] When the mutation is defective
Figure JPOXMLDOC01-appb-I000003
　図Ｓ３は、ある遺伝子に関し、野生型の二本鎖核酸の一方の鎖と、変異型の二本鎖核酸の一方の鎖であって、塩基配列Ｘ１と塩基配列Ｘ２の間の塩基配列Ｙが欠損している変異を有する場合を図示したものである。
　図Ｓ３において「変異配列」とは、連続するＸ１とＸ２の塩基配列を表す。
　「変異に対応する野生型の塩基配列」とは、野生型の核酸のＹの塩基配列を表す。
　「変異を含む領域」とは、変異を有する核酸鎖の、変異配列を含む塩基配列の領域（＊）を表す。
　「変異に対応する（野生型の）塩基配列を含む領域」とは、野生型の核酸の、Ｙを含む塩基配列の領域（＋）を表す。 FIG. S3 shows one strand of a wild-type double-stranded nucleic acid and one strand of a mutant-type double-stranded nucleic acid for a gene, wherein the nucleotide sequence Y between the nucleotide sequence X1 and the nucleotide sequence X2 is It is a diagram illustrating the case of having a missing mutation.
In FIG. S3, the “mutant sequence” represents the consecutive base sequences of X1 and X2.
“Wild-type nucleotide sequence corresponding to mutation” refers to the Y nucleotide sequence of the wild-type nucleic acid.
The term “mutation-containing region” refers to a region (*) of a base sequence containing a mutant sequence in a nucleic acid chain having a mutation.
A “region containing a (wild-type) base sequence corresponding to a mutation” refers to a region (+) of a base sequence containing Y in a wild-type nucleic acid.
　核酸としては、DNA及びRNAが挙げられ、好ましくはDNAである。本明細書において、DNAを構成する塩基を、本発明の分野で通常用いられる略号（アデニンを「Ａ」又は「a」、グアニンを「Ｇ」又は「g」、シトシンを「Ｃ」又は「c」、チミンを「Ｔ」又は「t」）で示す場合がある。 Nucleic acids include DNA and RNA, preferably DNA. In this specification, the abbreviations commonly used in the field of the present invention (adenine "A" or "a", guanine "G" or "g", cytosine "C" or "c") ”, and thymine as “T” or “t”).
＜１．本発明のプライマー対＞
　本発明のプライマー対は、
「３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がホスホロチオエート化（Ｓ化）されているフォワードプライマーと、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対」である。 <1. Primer pair of the present invention>
The primer pair of the present invention is
"A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is phosphorothioated (S-formed), and the 3' end A pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base to the 5' side is S-converted".
　以下、「フォワードプライマー」を「Ｆプライマー」と記載する。「リバースプライマー」を「Ｒプライマー」と記載する。 "Forward primer" is hereinafter referred to as "F primer". "Reverse primer" is described as "R primer".
　本発明のプライマー対に係るＦプライマー及びＲプライマーは、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合部分のリン酸残基の２個の酸素原子のうちの一つが硫黄原子に置換された、所謂ホスホロチオエート化（本明細書において「Ｓ化」とも記載する）されたものである。 The F primer and R primer according to the primer pair of the present invention are composed of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side of the phosphodiester bond moiety on the 3' side. One of the two oxygen atoms of the residue is substituted with a sulfur atom, so-called phosphorothioated (also described herein as “S”).
　本発明に係るプライマーのＳ化されたヌクレオチドの数は、変異判定の容易さの点から、１～４個が好ましく、２～４個がより好ましく、３個が特に好ましい。 From the viewpoint of ease of mutation determination, the number of S-modified nucleotides in the primer according to the present invention is preferably 1 to 4, more preferably 2 to 4, and particularly preferably 3.
　また、本発明のプライマー対において、ＦプライマーのＳ化されたヌクレオチドの数とＲプライマーのＳ化されたヌクレオチドの数は同じであっても異なっていてもよいが、同じであることが好ましい。 In addition, in the primer pair of the present invention, the number of S-nucleotides in the F primer and the number of S-nucleotides in the R primer may be the same or different, but are preferably the same.
　本発明に係るプライマーの長さとしては、プライマー配列としての特異性を維持するために必要な塩基数と考えられている１０塩基以上が好ましく、２０塩基以上の長さが更に好ましい。例えば２０～６０塩基が挙げられる。 The length of the primer according to the present invention is preferably 10 bases or more, which is considered to be the number of bases required to maintain specificity as a primer sequence, and more preferably 20 bases or more. Examples include 20 to 60 bases.
　ホスホロチオエート結合を導入したＳ化プライマーを合成する方法は周知の方法で行えばよく、例えばＤＮＡ合成装置でプライマーとなるオリゴヌクレオチドを合成するときに公知の方法によって必要な部位にホスホロチオエート(Phosphorothioate)結合を導入すればよい。
　例えばホスホルアミダイト(Phosphoramidite) 法でＤＮＡを合成する場合、適当なＳ化試薬（ホスホロチオエート化試薬）によって酸化処理を行うことで、通常のリン酸ジエステル結合ではなくホスホロチオエート結合をオリゴヌクレオチドに導入することができる。Ｓ化試薬としては、Beaucageの試薬(３Ｈ－１,２－ベンゾジチオール－３－オン１,１－ジオキシド)、TETD/Acetonitrile(TETD:tetraethylthiuram disulfide) 等が知られている。
　また、Ｓ化プライマーは、業者のカスタムサービスを利用して入手することもできる。 A well-known method may be used to synthesize an S-primer into which a phosphorothioate bond has been introduced. For example, when synthesizing oligonucleotides to serve as primers using a DNA synthesizer, a phosphorothioate bond is added to the required site by a known method. should be introduced.
For example, when synthesizing DNA by the phosphoramidite method, a phosphorothioate bond is introduced into the oligonucleotide instead of a normal phosphodiester bond by performing an oxidation treatment with an appropriate S-conjugation reagent (phosphorothioate reagent). can be done. Beaucage's reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide), TETD/Acetonitrile (TETD: tetraethylthiuram disulfide), and the like are known as sulfide reagents.
S-primers are also available through the custom service of vendors.
　本発明の好ましいプライマー対としては、例えば下記のプライマー対Ｍ及びプライマー対Ｗが挙げられる。 Preferred primer pairs of the present invention include, for example, primer pair M and primer pair W below.
　プライマー対Ｍ：
変異を有する二本鎖核酸の一方の鎖（第２鎖）にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第２鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチド３'側のホスホジエステル結合がＳ化されているＦプライマーと、
上記変異を有する二本鎖核酸のもう一方の鎖（第１鎖）にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第１鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーのプライマー対、
　プライマー対Ｗ：
野生型の二本鎖核酸の一方の鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
野生型の二本鎖核酸のもう一方の鎖（野生型の第１鎖）にアニールするプライマーであり、上記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーのプライマー対。 Primer pair M:
A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side. an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides from the second base from the 3′ end toward the 5′ side is S-converted;
A primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted,
Primer pair W:
A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the base sequence in the wild-type second strand, and placing a complementary sequence of the base sequence in the wild-type second strand corresponding to the mutation in the mutant second strand on the 3′ end side an F primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed,
A primer that anneals to the other strand of the wild-type double-stranded nucleic acid (wild-type first strand), and for mutation of the first strand of the mutant double-stranded nucleic acid (mutant-type first strand) Annealing to the region containing the base sequence in the corresponding wild-type first strand, and 3′ the complementary sequence of the base sequence in the wild-type first strand corresponding to the mutation in the mutant first strand An R primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side, which is on the terminal side and is S-ified. primer pair.
　本発明のプライマー対Ｍ及びプライマー対Ｗにおける「二本鎖核酸」としては、DNAが挙げられる。また、RNAとその相補鎖、RNAを鋳型とした逆転写反応を行って得られたcDNAとその相補鎖が挙げられる。 The "double-stranded nucleic acid" in primer pair M and primer pair W of the present invention includes DNA. Also included are RNA and its complementary strand, and cDNA obtained by reverse transcription using RNA as a template and its complementary strand.
　本発明のプライマー対Ｍ及びプライマー対Ｗにおいて、「塩基配列の変異を有する二本鎖核酸の一方の鎖（第２鎖）」及び「野生型の二本鎖核酸の一方の鎖（第２鎖）」に係る「一方の鎖（第２鎖）」との記載、及び「変異を有する二本鎖核酸のもう一方の鎖（第１鎖）」及び「野生型の二本鎖核酸のもう一方の鎖（第１鎖）」に係る「もう一方の鎖（第１鎖）」との記載は、単に二本鎖核酸の一方の鎖ともう一方の鎖を指すという意味であって、第１鎖及び第２鎖それぞれは、特段の意味を持たない。 In the primer pair M and primer pair W of the present invention, "one strand (second strand) of a double-stranded nucleic acid having a nucleotide sequence mutation" and "one strand (second strand) of a wild-type double-stranded nucleic acid )”, the description of “one strand (second strand)”, and “the other strand of the double-stranded nucleic acid having a mutation (first strand)” and “the other side of the wild-type double-stranded nucleic acid The description of "the other strand (first strand)" in relation to "the strand (first strand)" simply means that it refers to one strand and the other strand of a double-stranded nucleic acid, and the first Strand and secondary strand each have no particular meaning.
　本発明のプライマー対Ｍ及びプライマー対Ｗにおいて、「二本鎖核酸の一方の鎖（第２鎖）にアニールするプライマー」とは、そのプライマーがアニールする第２鎖の領域の相補鎖（第１鎖）の塩基配列と同じ塩基配列を、そのプライマーが有することを意味する。 In primer pair M and primer pair W of the present invention, "a primer that anneals to one strand (second strand) of a double-stranded nucleic acid" means a complementary strand (first This means that the primer has the same base sequence as the base sequence of the (strand).
　本発明のプライマー対Ｍ及びプライマー対Ｗにおいて、「二本鎖核酸のもう一方の鎖（第１鎖）にアニールするプライマー」とは、そのプライマーがアニールする第１鎖の領域の相補鎖（第２鎖）の塩基配列と同じ塩基配列を、そのプライマーが有することを意味する。 In the primer pair M and primer pair W of the present invention, the "primer that anneals to the other strand (first strand) of the double-stranded nucleic acid" means the complementary strand (first strand) of the region of the first strand to which the primer anneals. It means that the primer has the same base sequence as the base sequence of the second strand).
　本発明のプライマー対Ｍ及びプライマー対Ｗにおいて、「３’末端側」とは、３’末端ヌクレオチドを含む数個のヌクレオチドを含む領域、好ましくは１～４個のヌクレオチドを含む領域を指す。 In primer pair M and primer pair W of the present invention, the "3' terminal side" refers to a region containing several nucleotides including the 3' terminal nucleotide, preferably a region containing 1 to 4 nucleotides.
　本発明のプライマー対Ｍに係るＦプライマーは、３’末端ヌクレオチドを含む数個、好ましくは１～４個、より好ましくは１～３個、更に好ましくは１個のヌクレオチドの塩基配列が塩基配列の変異を有する二本鎖核酸の第２鎖の変異配列の相補配列を有するものであることが好ましい。３’末端ヌクレオチドの塩基が上記第２鎖の変異塩基の相補塩基であることが特に好ましい。 The F primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3′ terminal nucleotide of the base sequence. It preferably has a sequence complementary to the mutated sequence of the second strand of the mutated double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the mutated base of the second strand.
　本発明のプライマー対Ｍに係るＲプライマーは、３’末端ヌクレオチドを含む数個、好ましくは１～４個、よりに好ましくは１～３個、更に好ましくは１個のヌクレオチドの塩基配列が第１鎖の変異配列の相補配列を有するものであることが好ましい。３’末端ヌクレオチドの塩基が第１鎖の変異塩基の相補塩基であることが特に好ましい。 The R primer associated with the primer pair M of the present invention has a base sequence of several, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 nucleotides including the 3' terminal nucleotide. Preferably, it has the complementary sequence of the mutant sequence of the strand. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the mutated base of the first strand.
　本発明のプライマー対Ｗに係るＦプライマーは、その３’末端ヌクレオチドを含む数個、好ましくは１～４個、より好ましくは１～３個、更に好ましくは１個のヌクレオチドの塩基が、変異を有する二本鎖核酸の第２鎖の変異に対応する野生型の二本鎖核酸の第２鎖中の塩基配列の相補配列を有するものであることが好ましい。３’末端ヌクレオチドの塩基が上記変異に対応する上記野生型の二本鎖核酸の第２鎖中の塩基の相補塩基であることが特に好ましい。 In the F primer according to the primer pair W of the present invention, several, preferably 1 to 4, more preferably 1 to 3, more preferably 1 nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the second strand of the wild-type double-stranded nucleic acid that corresponds to the mutation in the second strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the second strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
　本発明のプライマー対Ｗに係るＲプライマーは、その３’末端ヌクレオチドを含む数個、好ましくは１～４個、より好ましくは１～３個、更に好ましくは１個のヌクレオチドの塩基が、変異を有する二本鎖核酸の第１鎖の変異に対応する野生型の二本鎖核酸の第１鎖中の塩基配列の相補配列を有するものであることが好ましい。３’末端ヌクレオチドの塩基が上記変異に対応する上記野生型の二本鎖核酸の第１鎖中の塩基の相補塩基であることが特に好ましい。 In the R primer associated with the primer pair W of the present invention, several, preferably 1 to 4, more preferably 1 to 3, more preferably 1 nucleotide bases including the 3' terminal nucleotide are mutated. It preferably has a sequence complementary to the nucleotide sequence in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation in the first strand of the double-stranded nucleic acid. It is particularly preferred that the base of the 3' terminal nucleotide is the complementary base of the base in the first strand of the wild-type double-stranded nucleic acid corresponding to the mutation.
　本発明の上記プライマー対Ｍは、被検試料の核酸の塩基配列が変異を持つ場合に、このプライマー対を用いた核酸増幅反応で増幅産物が得られるが、被検試料の核酸の塩基配列が変異を持たない場合には、このプライマー対を用いた核酸増幅反応で増幅産物が得られない、プライマー対である。すなわち、本発明のプライマー対Ｍは変異検出用プライマー対である。 The above-described primer pair M of the present invention provides an amplification product in a nucleic acid amplification reaction using this primer pair when the nucleotide sequence of the nucleic acid of the test sample has a mutation. A primer pair that does not have a mutation does not yield an amplification product in a nucleic acid amplification reaction using this primer pair. That is, the primer pair M of the present invention is a primer pair for mutation detection.
　一方、本発明の上記プライマー対Ｗは、被検試料の核酸の塩基配列が野生型である場合に、このプライマー対を用いた核酸増幅反応で増幅産物が得られるプライマー対である。すなわち、本発明のプライマー対Ｗは、検出対象の変異に関して野生型である、野生型検出用プライマー対である。 On the other hand, the primer pair W of the present invention is a primer pair that gives an amplification product in a nucleic acid amplification reaction using this primer pair when the base sequence of the nucleic acid in the test sample is of the wild type. That is, the primer pair W of the present invention is a wild-type detection primer pair that is wild-type with respect to the mutation to be detected.
　本発明の上記プライマー対Ｍ及びプライマー対Ｗについて、それぞれ以下に詳説する。 The primer pair M and primer pair W of the present invention will be described in detail below.
（１）本発明のプライマー対Ｍについて (1) Primer pair M of the present invention
　本発明のプライマー対Ｍの例としては、塩基配列の置換又は挿入の変異を検出するプライマー対（Ｍ－１）、又は塩基の欠損の変異を検出するプライマー対（Ｍ－２）が挙げられる。 Examples of the primer pair M of the present invention include a primer pair (M-1) for detecting nucleotide sequence substitution or insertion mutations, or a primer pair (M-2) for detecting nucleotide deletion mutations.
１）本発明のプライマー対（Ｍ－１）
　塩基配列の置換又は挿入を検出する本発明のプライマー対（Ｍ－１）は、以下のものである。 1) Primer pair (M-1) of the present invention
The primer pair (M-1) of the present invention for detecting nucleotide sequence substitution or insertion is as follows.
　置換又は挿入変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第２鎖の上記変異配列の相補配列を３’末端側に有し、その他の塩基配列は上記第２鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記変異を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、上記第１鎖の変異配列の相補配列を３’末端側に有し、その他の塩基配列は上記第１鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーのプライマー対。 A primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation, anneals to the region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side, The other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the mutation of the second strand, and is continuous from the second base from the 3'-end toward the 5'-side. an F primer in which the phosphodiester bond on the 3′ side of four nucleotides is S-converted;
A primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and is continuous from the second base from the 3' end toward the 5' side. A primer pair of R primers in which the phosphodiester bond on the 3' side of the nucleotide is S-sylated.
　検出対象の塩基配列の置換又は挿入の変異は、１～４塩基の置換又は挿入が好ましい。１～３塩基の置換又は挿入が好ましい。１塩基の置換又は挿入が更に好ましい。 Substitution or insertion mutations in the base sequence to be detected are preferably substitutions or insertions of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
　本発明のプライマー対Ｍ－１としては、以下のものが好ましい。
　置換又は挿入変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、３’末端ヌクレオチドを含む連続する１～４個のヌクレオチドは上記第２鎖の変異配列の相補配列と同じであって、その他の塩基配列は上記第２鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記変異を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、３'末端ヌクレオチドを含む連続する１～４個のヌクレオチドは上記第１鎖の上記変異配列の相補配列と同じであって、その他の塩基配列は上記第１鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 As the primer pair M-1 of the present invention, the following are preferable.
A primer that anneals to the second strand of a double-stranded nucleic acid having a substitution or insertion mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide of the second strand It is the same as the complementary sequence of the mutant sequence, and the other base sequence is the same as the complementary sequence of the 3' side base sequence adjacent to the mutation in the second strand, from the second base from the 3' end an F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides toward the 5′ side is S-formed;
A primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and 1 to 4 consecutive nucleotides including the 3' terminal nucleotide are the mutation of the first strand It is the same as the complementary sequence of the sequence, and the rest of the nucleotide sequence is the same as the complementary sequence of the 3′ side nucleotide sequence adjacent to the mutation of the first strand, and 5 from the second base from the 3′ end Primer pairs of R primers in which the phosphodiester bond on the 3' side of 1-4 consecutive nucleotides toward the ' side is S-merified.
　本発明のプライマー対Ｍ－１としては、以下のものがより好ましい。
　一塩基の置換又は挿入変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、３’末端ヌクレオチドは上記第２鎖の上記変異塩基の相補塩基と同じであって、その他の塩基配列は上記第２鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記変異を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記変異を含む領域にアニールし、３'末端ヌクレオチドは上記第１鎖の上記変異塩基の相補塩基と同じであって、その他の塩基配列は上記第１鎖の上記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 As the primer pair M-1 of the present invention, the following are more preferable.
A primer that anneals to the second strand of a double-stranded nucleic acid having a single-base substitution or insertion mutation, anneals to the region containing the mutation, and has a 3′ terminal nucleotide that is complementary to the mutated base of the second strand. The other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, from the second base from the 3' end to the 5' side An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed,
A primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and has the same 3′ terminal nucleotide as the complementary base of the mutated base of the first strand, The other nucleotide sequence is the same as the complementary sequence of the 3′ side nucleotide sequence adjacent to the mutation of the first strand, and is continuous from the second base from the 3′ end toward the 5′ side. A primer pair of R primers in which the phosphodiester bonds on the 3′ sides of the four nucleotides are S-sylated.
　詳細は、後記する図Ａ及び図Ｂを用いて説明する。 Details will be explained using Figures A and B below.
（ｉ）置換検出用プライマー対（Ｍ－１）について (i) For displacement detection primer pair (M-1)
　塩基配列の置換を検出する本発明のプライマー対（Ｍ－１）について、下記図Ａをもとに説明する。図Ａでは、各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 The primer pair (M-1) of the present invention for detecting base sequence substitution will be explained based on Figure A below. FIG. A illustrates the case where the number of S-nucleotides in each primer is three.
　尚、後記する図Ａ～図Ｄ、及び図Ａ２～図Ｄ２に記載された塩基配列は、本発明のプライマー対を説明するための模式的な塩基配列である。 The base sequences shown in Figures A to D and Figures A2 to D2 described below are schematic base sequences for explaining the primer pairs of the present invention.
　また、各図Ａ～図Ｄ、及び図Ａ２～図Ｄ２において、Ｓ化されている塩基は太線で囲んで示す。更に、ＦはＦプライマーを、ＲはＲプライマーを示す。 In addition, in each of Figures A to D and Figures A2 to D2, S-formed bases are shown surrounded by thick lines. Furthermore, F indicates the F primer and R indicates the R primer.
　更に、各図Ａ～図Ｄ、及び図Ａ２～図Ｄ２の説明において塩基配列について説明する場合であって特にその方向を示していない場合は、常法に従い、５’側から３’側に向かっての塩基配列を示す。 Furthermore, in the description of each of Figures A to D and Figures A2 to D2, when the base sequence is described and the direction is not particularly indicated, the sequence is from the 5' side to the 3' side in accordance with the usual method. all base sequences are shown.
[図Ａ]
Figure JPOXMLDOC01-appb-I000004
[Figure A]
Figure JPOXMLDOC01-appb-I000004
図Ａ(１)：野生型の二本鎖核酸の第１鎖と第２鎖の塩基配列を図示したものである。 FIG. A(1): A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
図Ａ(２)：１塩基置換検出用プライマー対
　図Ａ(２)は、野生型の塩基配列に対し●印の塩基が、第１鎖ではグアニン(g)からアデニン(a)に置換されており、第２鎖ではシトシン(c)からチミン(t)に置換されている１塩基置換の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖の変異塩基(t)の相補塩基であるaを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記変異塩基であるtに隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の変異塩基(a)の相補塩基であるtを、その３’末端に有する。そしてその他の塩基配列は、上記第１鎖の上記変異塩基であるaに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A (2): Primer pair for detecting single base substitution Figure A (2) shows that the bases marked ● in the wild-type base sequence are substituted from guanine (g) to adenine (a) in the first strand. An example of a primer pair (M-1) for detecting a single-base substitution mutation in which cytosine (c) is substituted with thymine (t) in the second strand is shown.
The F primer has at its 3′ end a, the complementary base of the mutated base (t) of the second strand. The other nucleotide sequences are the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the mutated nucleotide t of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer has t at its 3' end, which is the complementary base of the mutated base (a) of the first strand. The other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide a of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ａ(３)：２塩基置換検出用プライマー対
　図Ａ(３)は、野生型の塩基配列に対し、●印の塩基配列が、第１鎖ではtgからcaに置換されており、第２鎖ではcaからtgに置換されている２塩基置換の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖の変異配列(tg)の相補配列であるcaを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記変異配列であるtgに隣接する３’側の塩基配列（ccaatcaca）の相補配列(tgtgattgg)と同じである。また、このＦプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（cgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の変異配列(ca)の相補配列であるtgを、その３’末端に有する。そしてその他の塩基配列は、上記第１鎖の上記変異配列であるcaに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A (3): Primer pair for detecting double-base substitution An example of a primer pair (M-1) that detects a 2-base mutation in which the chain is substituted from ca to tg is shown.
The F primer has at its 3' end ca, the complementary sequence of the mutated sequence (tg) of the second strand. The other nucleotide sequence is the same as the complementary sequence (tgtgattgg) of the 3'-side nucleotide sequence (ccaatcaca) adjacent to the mutated sequence tg of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgg) from the second base from the 3' end of the F primer toward the 5' side (cgg) is S-formed. .
The R primer has at its 3' end tg, the complementary sequence of the mutant sequence (ca) of the first strand. The other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to ca, which is the mutant sequence of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
図Ａ(４)：３塩基置換検出用プライマー対
　図Ａ(４)は、野生型の塩基配列に対し、●印の塩基配列が、第１鎖ではgtgからtcaに置換されており、第２鎖ではcacからtgaに置換されている３塩基置換の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖の変異配列(tga)の相補配列であるtcaを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記変異配列であるtgaに隣接する３’側の塩基配列（caatcaca）の相補配列（tgtgattg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（ctg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の変異配列(tca)の相補配列であるtgaを、その３’末端に有する。そしてその他の塩基配列は、上記第１鎖の上記変異配列であるtcaに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gtg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A (4): Primer pair for detecting 3 base substitutions An example of a primer pair (M-1) that detects a 3-base substitution mutation in which cac is replaced with tga in the strand is shown.
The F primer has at its 3' end tca, the complement of the second strand mutant sequence (tga). The other nucleotide sequence is the same as the complementary sequence (tgtgattg) of the 3′-side nucleotide sequence (caatcaca) adjacent to the mutated sequence tga of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed. .
The R primer has at its 3' end tga, the complementary sequence of the first strand mutant sequence (tca). The other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated sequence tca of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
(ii)挿入検出用プライマー対（Ｍ－１）について
　塩基配列の挿入を検出する本発明のプライマー対Ｍ－１について、下記図Ｂをもとに説明する。
　図Ｂでは、各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 (ii) Insertion Detection Primer Pair (M-1) The primer pair M-1 of the present invention for detecting insertion of a nucleotide sequence will be described with reference to the following diagram B. FIG.
FIG. B exemplifies the case where the number of S-nucleotides in each primer is three.
[図Ｂ]
Figure JPOXMLDOC01-appb-I000005
[Figure B]
Figure JPOXMLDOC01-appb-I000005
図Ｂ(１)：野生型の二本鎖核酸の第１鎖と第２鎖の塩基配列を図示したものである。 FIG. B(1): A representation of the base sequences of the first and second strands of a wild-type double-stranded nucleic acid.
図Ｂ(２)：１塩基挿入検出用プライマー対
　図Ｂ(２)は、野生型の塩基配列に対し、第１鎖では●印の位置にtが挿入されており、第２鎖では●印の位置にaが挿入されている１塩基挿入の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖に挿入された塩基(a)の相補塩基であるtを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記変異塩基であるaに隣接する３’側の塩基配列（caccaatcaca）の相補配列（tgtgattggtg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gtg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖に挿入された塩基(t)の相補塩基であるaを、その３’末端に有する。そしてその他の塩基配列は、上記第１鎖の上記変異塩基であるtに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B (2): Primer pair for detecting single base insertion Figure B (2) shows that t is inserted at the position marked ● in the first strand and marked ● in the second strand against the wild-type base sequence. An example of a primer pair (M-1) for detecting a single-base insertion mutation in which a is inserted at the position of is shown.
The F primer has t at its 3' end, which is the complementary base of base (a) inserted in the second strand. The other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the 3'-side nucleotide sequence (caccaatcaca) adjacent to the mutated nucleotide a of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base g from the 3' end of the F primer toward the 5' side is S-formed. .
The R primer has at its 3' end a, the complementary base of the base (t) inserted in the first strand. The other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutated nucleotide t of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ｂ(３)：２塩基挿入検出用プライマー対
　図Ｂ(３)は、野生型の塩基配列に対し、第１鎖では●印の位置にtaが挿入されており、第２鎖では●印の位置にtaが挿入されている２塩基挿入の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖に挿入されたtaの相補配列であるtaを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記変異配列であるtaに隣接する３’側の塩基配列（caccaatcaca）の相補配列（tgtgattggtg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖に挿入されたtaの相補配列であるtaを、その３’末端に有する。そしてその他の塩基配列は、上記第１鎖の上記変異配列であるtaに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B (3): Primer pair for detecting double base insertion Figure B (3) shows that ta is inserted at the position marked ● in the first strand and marked ● in the second strand against the wild-type base sequence. An example of a primer pair (M-1) for detecting a 2-base insertion mutation in which ta is inserted at the position of is shown.
The F primer has at its 3' end ta, the complement of ta inserted in the second strand. The other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the mutant sequence ta of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the F primer toward the 5' side (tgt) is S-formed. .
The R primer has at its 3' end ta, the complementary sequence of ta inserted in the first strand. The other nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to the mutant sequence ta of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgt) from the second base from the 3' end of the R primer toward the 5' side (tgt) is S-formed. .
図Ｂ(４)：３塩基挿入検出用プライマー対
　図Ｂ(４)は、野生型の塩基配列に対し、第１鎖では●印の位置にtagが挿入されており、第２鎖では●印の位置にctaが挿入されている３塩基挿入の変異を検出するプライマー対（Ｍ－１）の例を示す。
　Ｆプライマーは、第２鎖に挿入されたctaの相補配列であるtagを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記変異配列であるctaに隣接する３’側の塩基配列（caccaatcaca）の相補配列（tgtgattggtg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（atg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖に挿入されたtagの相補配列であるctaを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記変異配列であるtagに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tcg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B (4): Primer pair for detecting 3-base insertion In Figure B (4), a tag is inserted at the position marked ● in the first strand against the wild-type base sequence, and marked ● in the second strand. An example of a primer pair (M-1) for detecting a 3-base insertion mutation in which cta is inserted at the position of is shown.
The F primer has tag at its 3' end, which is the complementary sequence of cta inserted in the second strand. The other nucleotide sequences are the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the mutant sequence cta of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (atg) from the second base from the 3' end of the F primer toward the 5' side (atg) is S-formed. .
The R primer has cta at its 3' end, which is the complementary sequence of the tag inserted in the first strand. The other nucleotide sequences are the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to tag, which is the mutant sequence of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tcg) from the second base from the 3' end of the R primer toward the 5' side (tcg) is S-formed. .
　本発明のプライマー対Ｍ－１の具体例として、例えばヒトのα１アンチトリプシン遺伝子（ゲノム DNA GenBank Accession No. NG_008290.1、mRNA GenBank Accession No. NM_000295.5）の１塩基置換変異であるPiS変異の変異検出用プライマー対（本発明のプライマー対Ｍ－１、上記図Ａ（２）の場合に相当する。）を例にとり、以下に説明する。 As a specific example of the primer pair M-1 of the present invention, for example, PiS mutation, which is a single base substitution mutation of human α1-antitrypsin gene (genomic DNA GenBank Accession No. NG_008290.1, mRNA GenBank Accession No. NM_000295.5) The mutation detection primer pair (primer pair M-1 of the present invention, corresponding to the case of FIG. A(2) above) will be described below as an example.
　ヒトのα１アンチトリプシンの遺伝子変異であるPiS変異は、以下の１塩基置換である。
　　　　PiS：c.863A>T p.Glu288Val (Glu264Val) A PiS mutation, which is a gene mutation of human α1-antitrypsin, is the following single base substitution.
PiS: c.863A>T p.Glu288Val (Glu264Val)
　即ちヒトのPiS変異はcoding DNAの863番目のアデニン(A)がチミン(T)に置換された１塩基置換であり、その結果α１アンチトリプシンのアミノ酸配列の288番目のGluがValに置換された変異である。
　この１塩基置換の検出を対象とする本発明のプライマー対を設計するにあたり、α１アンチトリプシンの遺伝子の二本鎖DNAの、AがTに置換された方の鎖を第１鎖とする。上記二本鎖DNAの、同じ部分の塩基のTがAに置換された方の鎖を、第２鎖とする。 That is, the human PiS mutation is a single-base substitution in which the 863rd adenine (A) in the coding DNA is replaced with thymine (T), and as a result, the 288th Glu in the α1-antitrypsin amino acid sequence is replaced with Val. Mutation.
In designing the primer pair of the present invention for detecting this single base substitution, the strand in which A is replaced with T in the double-stranded DNA of the α1-antitrypsin gene is defined as the first strand. The strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
　尚、本明細書で記載する各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 In addition, in the base sequences of each primer described in this specification, the * mark indicates the position where the phosphodiester bond is S-converted.
PiS変異型検出用プライマー対（Ｍ－１）としては、例えば下記のものが設計される。 As the PiS mutation detection primer pair (M-1), for example, the following are designed.
i)１塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCTGG*T-3’（配列番号２９）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCATTT*A-3’（配列番号３０） i) 1 base S primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCTGG*T-3' (SEQ ID NO: 29)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCATTT*A-3' (SEQ ID NO: 30)
ii)２塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCTG*G*T-3’（配列番号３３）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCATT*T*A-3’（配列番号３４）
iii)３塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*T-3’（配列番号３７）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3’（配列番号３８） ii) 2-base S-primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCTG*G*T-3' (SEQ ID NO: 33)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCATT*T*A-3' (SEQ ID NO: 34)
iii) 3-base S-primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*T-3' (SEQ ID NO: 37)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3' (SEQ ID NO: 38)
　PiS変異型検出用プライマー対のＦプライマーは、PiS変異を有する二本鎖核酸の第２鎖の変異塩基(A)の相補塩基であるTを、３'末端に有する。その他の塩基配列は、上記第２鎖の変異塩基(A)に隣接する３’側の塩基配列の相補配列と同じになるように設計される。
　PiS変異型検出用プライマー対のＲプライマーは、PiS変異を有する２本鎖核酸の第１鎖の塩基(T)の相補塩基であるAを、３'末端に有する。その他の塩基配列は、上記第１鎖の変異塩基(T)に隣接する３’側の塩基配列と同じになるように設計される。
　更に上記のプライマー対の例では３'末端から２～４番目のヌクレオチドは、そのホスホジエステル結合がＳ化されたヌクレオチドである。 The F primer of the PiS mutation detection primer pair has T at the 3′ end, which is the complementary base of the mutated base (A) of the second strand of the double-stranded nucleic acid having the PiS mutation. Other nucleotide sequences are designed to be the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the mutated nucleotide (A) of the second strand.
The R primer of the pair of primers for detecting PiS mutation type has, at its 3' end, A, which is the complementary base to the base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation. Other nucleotide sequences are designed to be the same as the 3' nucleotide sequence adjacent to the mutated nucleotide (T) of the first strand.
Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
　PiS変異型検出用プライマー対の設計方法の一例を以下に説明する。
　Ｆプライマーは、その３'末端塩基はPiS遺伝子の変異塩基(T)と同じであり、その他の塩基配列は、PiS変異を有する２本鎖核酸の第１鎖の上記変異塩基(T)より５’側に向けての塩基配列と同じになるように設計される。
　一方Ｒプライマーは、その３'末端塩基はPiS遺伝子の変異塩基(T)の相補塩基(A)と同じであり、その他の塩基配列は、PiS変異を有する２本鎖核酸の第１鎖の上記変異塩基(T)に隣接する３’側に向けての塩基配列の相補配列と同じになるように設計される。
　更に上記のプライマー対の例では３'末端から２～４番目のヌクレオチドは、そのホスホジエステル結合がＳ化されたヌクレオチドとする。 An example of a method for designing a primer pair for detecting PiS mutation is described below.
The F primer has the same 3′ terminal base as the mutated base (T) of the PiS gene, and the other base sequence is 5 from the mutated base (T) of the first strand of the double-stranded nucleic acid having the PiS mutation. It is designed to be the same as the base sequence facing the ' side.
On the other hand, the R primer has the same 3′ terminal base as the complementary base (A) of the mutated base (T) of the PiS gene, and the other base sequence is the same as the first strand of the double-stranded nucleic acid having the PiS mutation. It is designed to be the same as the complementary sequence of the base sequence towards the 3' side adjacent to the mutated base (T).
Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
　本発明のプライマー対Ｍのその他の例としては、例えばヒトα１アンチトリプシンの別の遺伝子変異であるPiZ変異（１塩基変異、c.1096G>A p.Glu366Lys）を検出する、下記の変異検出用プライマー対が挙げられる。 Other examples of the primer pair M of the present invention include the following mutation detection for detecting PiZ mutation (single nucleotide mutation, c.1096G>A p.Glu366Lys), which is another genetic mutation of human α1-antitrypsin. Primer pairs are included.
i)１塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCGAC*A-3’ （配列番号４３）
　Ｒプライマー：5’-CCCCAGCAGCTTCAGTCCCTTTCT*T-3’ （配列番号４４） i) 1 base S primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCGAC*A-3' (SEQ ID NO: 43)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTTCT*T-3' (SEQ ID NO: 44)
ii)２塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCGA*C*A-3’ （配列番号４７）
　Ｒプライマー：5’-CCCCAGCAGCTTCAGTCCCTTTC*T*T-3’ （配列番号４８） ii) 2-base S-primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCGA*C*A-3' (SEQ ID NO: 47)
R primer: 5'-CCCCAGCAGCTTTCAGTCCCTTTC*T*T-3' (SEQ ID NO: 48)
iii)３塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCG*A*C*A-3’ （配列番号５１）
　Ｒプライマー：5’-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3’ （配列番号５２） iii) 3-base S-primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*A-3' (SEQ ID NO: 51)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3' (SEQ ID NO: 52)
　本発明のプライマー対Ｍ－１のその他の例としては、例えば下記表１に記載のものが挙げられる。 Other examples of the primer pair M-1 of the present invention include those listed in Table 1 below.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
[変異の位置]
BRAF：c1799T＞A(V600E
PiS：c.863A>T p.Glu288Val
PiZ：c.1096G>A p.Glu366Lys
PKD1：c. 9864C>A
PKLR：c. 693 + 304G> A 
VPS13B：g.4411950_4411953delGTTT
N501Y：A23063T
E484K：G23012A [Position of mutation]
BRAF: c1799T > A (V600E
PiS: c.863A>T p.Glu288Val
PiZ: c.1096G>A p.Glu366Lys
PKD1: c.9864C>A
PKLR: c.693+304G>A
VPS13B: g.4411950_4411953delGTTT
N501Y: A23063T
E484K: G23012A
２）本発明のプライマー対（Ｍ－２）
　塩基配列の欠損を検出する本発明のプライマー対（Ｍ－２）は、以下のものである。
　欠損変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、上記欠損を含む領域にアニールし、上記第２鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記第２鎖の上記欠損に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記欠損を有する二本鎖核酸の第１鎖にアニールするプライマーであり、上記欠損を含む領域にアニールし、上記第１鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記第１鎖の上記欠損に隣接する３’側の塩配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 2) Primer pair (M-2) of the present invention
The primer pair (M-2) of the present invention for detecting nucleotide sequence defects is as follows.
A primer that anneals to the second strand of a double-stranded nucleic acid having a deletion mutation, anneals to the region containing the deletion, and anneals to the 5'-side nucleotide sequence adjacent to the deletion of the second strand. The other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, from the second base from the 3' end toward the 5' side An F primer in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-formed,
A primer that anneals to the first strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and attaches a sequence complementary to the 5' base sequence adjacent to the deletion of the first strand to the 3' end. The other base sequence is the same as the complementary sequence of the salt sequence on the 3' side adjacent to the deletion of the first strand, from the second base from the 3' end toward the 5' side A pair of R primers in which the phosphodiester bond on the 3′ side of 1 to 4 consecutive nucleotides of is S-converted.
　上記本発明のプライマー対（Ｍ－２）に係る「欠損を含む領域にアニールする」とは、少なくとも欠損位置の両端（５'側及び３'側）の塩基に結合することを意味する。 "Annealing to the deletion-containing region" of the primer pair (M-2) of the present invention means binding to at least bases on both ends (5' side and 3' side) of the deletion position.
　検出対象の塩基配列の欠損の変異は、１～４塩基の欠損が好ましい。１～３塩基の欠損がより好ましい。１塩基欠損が更に好ましい。 Deletion mutations in the base sequence to be detected are preferably deletions of 1 to 4 bases. A deletion of 1 to 3 bases is more preferred. A single base deletion is more preferred.
　詳細は、後記する図Ｃ及び図Ｄを用いて説明する。 Details will be explained using Figures C and D below.
（ｉ）１塩基欠損検出用プライマー対（Ｍ－２）について
　塩基配列の１塩基欠損を検出する本発明のプライマー対（Ｍ－２）について、下記図Ｃをもとに説明する。
　図Ｃでは、野生型の塩基配列に対し、矢印の位置の１塩基が欠損した変異を検出するプライマー対（Ｍ－２）の例を３例示す。第１鎖では、矢印のgが欠損しており、第２鎖では矢印のcが欠損している。
　図Ｃ（２）～図Ｃ（４）において、塩基配列の空欄は塩基が欠損していることを示し、実際には空欄の５'側の塩基と３’側の塩基は連続している。
　尚、図Ｃでは各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 (i) Primer pair (M-2) for detecting 1-base deletion The primer pair (M-2) of the present invention for detecting 1-base deletion in a base sequence will be described with reference to Figure C below.
FIG. C shows three examples of primer pairs (M-2) that detect mutations in which one base is deleted at the position of the arrow in the wild-type base sequence. The first strand lacks the arrow g and the second strand lacks the arrow c.
In Figures C(2) to C(4), blanks in the base sequences indicate that bases are missing, and in fact, the 5' and 3' bases of the blanks are continuous.
Note that FIG. C illustrates a case where the number of S-nucleotides in each primer is three.
[図Ｃ]
Figure JPOXMLDOC01-appb-I000007
[Figure C]
Figure JPOXMLDOC01-appb-I000007
図Ｃ(１)：野生型の第１鎖と第２鎖の塩基配列を図示したものである。 FIG. C(1): A representation of the base sequences of the wild-type first and second strands.
図Ｃ(２)：１塩基欠損検出用プライマー１
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基(g)の相補塩基であるcを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記欠損に隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基(t)の相補塩基であるaを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C (2): Primer 1 for detecting 1 base deletion
The F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ｃ(３)：１塩基欠損検出用プライマー２
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基配列(tg)の相補配列であるcaを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記欠損に隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（ctg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基配列(gt)の相補配列であるacを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（agt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C (3): Primer 2 for detecting 1 base deletion
The F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctg) from the second base from the 3' end of the F primer toward the 5' side (ctg) is S-formed. .
The R primer has at its 3' end ac, the complementary sequence of the 5' nucleotide sequence (gt) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
図Ｃ(４)：１塩基欠損検出用プライマー３
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基配列(atg)の相補配列であるcatを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記欠損に隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（act）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基配列(ggt)の相補配列であるaccを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（cag）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C (4): Primer 3 for detecting 1 base deletion
The F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
The R primer has at its 3' end acc, the complementary sequence of the 5' nucleotide sequence (ggt) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cag) from the second base from the 3' end of the R primer toward the 5' side (cag) is S-formed. .
（ii）４塩基欠損検出用プライマー対（Ｍ－２）について
　塩基配列の４塩基欠損を検出する本発明のプライマー対（Ｍ－２）について、下記図Ｄをもとに説明する。
　図Ｄでは、野生型の塩基配列に対し、●印の４塩基が欠損した変異を検出するプライマー対（Ｍ－２）の例を３例示す。第１鎖では、●印のggtgが欠損しており、第２鎖では、●印のcaccが欠損している。
　図Ｄ（２）～図Ｄ（４）において、塩基配列の空欄は塩基が欠損していることを示し、実際には空欄の５'側の塩基と３’側の塩基は連続している。
　尚、図Ｄでは各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 (ii) Primer pair (M-2) for detecting 4-base deletion The primer pair (M-2) of the present invention for detecting 4-base deletion in a nucleotide sequence will be described with reference to Figure D below.
FIG. D shows three examples of primer pairs (M-2) for detecting mutations in which 4 bases marked with ● are deleted from the wild-type base sequence. The first strand lacks ggtg marked with ●, and the second strand lacks cacc marked with ●.
In Figures D(2) to D(4), a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous.
Note that FIG. D illustrates a case where the number of S-nucleotides in each primer is three.
[図Ｄ]
Figure JPOXMLDOC01-appb-I000008
[Figure D]
Figure JPOXMLDOC01-appb-I000008
図Ｄ(１)：野生型の第１鎖と第２鎖の塩基配列を図示したものである。 FIG. D(1): A representation of the base sequences of the wild-type first and second strands.
図Ｄ(２)：４塩基欠損検出用プライマー対―１
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基(g)の相補塩基であるcを、その３’末端に有する。そしてその他の塩基配列は、上記第２鎖の上記欠損に隣接する３’側の塩基配列（aatcaca）の相補配列（tgtgatt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基(t)の相補塩基であるaを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure D (2): Primer pair-1 for detecting 4-base deletion
The F primer has c at its 3' end which is the complementary base of the 5' base (g) adjacent to the second strand defect. The rest of the base sequence is the same as the complementary sequence (tgtgatt) of the base sequence (aatcaca) on the 3' side adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tta) from the second base from the 3' end of the F primer toward the 5' side (tta) is S-formed. .
The R primer has at its 3' end a, the complementary base of the 5' base (t) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ｄ(３)：４塩基欠損検出用プライマー対―２
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基配列(tg)の相補配列であるcaを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記欠損に隣接する３’側の塩基配列（aatcaca）の相補配列（tgtgatt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（ctt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基配列(tt)の相補配列であるaaを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（agt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure D (3): Primer pair-2 for detecting 4-base deletion
The F primer has at its 3' end ca, the complementary sequence of the 5' nucleotide sequence (tg) adjacent to the second strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3′-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ctt) from the second base from the 3' end of the F primer toward the 5' side (ctt) is S-formed. .
The R primer has at its 3' end aa, which is the complementary sequence of the 5' nucleotide sequence (tt) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (agt) from the second base from the 3' end of the R primer toward the 5' side (agt) is S-formed. .
図Ｄ(４)：４塩基欠損検出用プライマー対―３
　Ｆプライマーは、第２鎖の欠損に隣接する５’側の塩基配列(atg)の相補配列であるcatを、その３’末端に有する。そしてその他の塩基配列は、第２鎖の上記欠損に隣接する３’側の塩基配列（aatcaca）の相補配列（tgtgatt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（act）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、第１鎖の欠損に隣接する５’側の塩基配列(att)の相補配列であるaatを、その３’末端に有する。そしてその他の塩基配列は、第１鎖の上記欠損に隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（aag）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure D (4): Primer pair for detecting 4-base deletion -3
The F primer has cat at its 3' end, which is the complementary sequence of the 5' nucleotide sequence (atg) adjacent to the second strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgatt) of the 3'-side nucleotide sequence (aatcaca) adjacent to the deletion of the second strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (act) from the second base from the 3' end of the F primer toward the 5' side (act) is S-formed. .
The R primer has at its 3' end aat, which is the complementary sequence of the 5' nucleotide sequence (att) adjacent to the first strand defect. The rest of the nucleotide sequence is the same as the complementary sequence (gcggatg) of the 3'-side nucleotide sequence (catccgc) adjacent to the deletion of the first strand. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (aag) from the second base from the 3' end of the R primer toward the 5' side (aag) is S-formed. .
　尚、特に図を用いて説明はしないが、２塩基欠損又は３塩基欠損を検出するプライマー対も、同様の方法で設計することができる。 Although not specifically described using the figures, a primer pair for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method.
　本発明のプライマー対Ｍ－２の具体例としては、例えば下記のものが挙げられる。 Specific examples of the primer pair M-2 of the present invention include the following.
　イヌVPS13B遺伝子の４塩基欠損が知られており、その変異型検出用プライマー対として、下記のプライマー対が設計される。 A 4-base deletion in the canine VPS13B gene is known, and the following primer pair is designed as a primer pair for detecting the mutation.
・VPS13B変異型検出用プライマー対
　Ｆプライマー：5’-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3’（配列番号７３）
　Ｒプライマー：5’-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3’（配列番号７４） ・ VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73)
R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3' (SEQ ID NO: 74)
　本発明のプライマー対Ｍ－２のその他の例としては、例えば下記表２に記載のものが挙げられる。 Other examples of the primer pair M-2 of the present invention include those listed in Table 2 below.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　一般にプライマー同士の相補になる領域が多いほどプライマーダイマーが形成される確率が高いことを考慮すると、本発明のプライマー対Ｍとしては、図Ａ(２)、図Ｂ(２)、図Ｃ(２)、及び図Ｄ(２)のプライマー対が好ましい。図Ａ(２)及び図Ｂ(２)のプライマー対がより好ましい。 Considering that the probability of primer dimer formation is generally higher as the number of complementary regions between primers increases, the primer pair M of the present invention is shown in FIG. ), and the primer pair of Figure D(2) are preferred. More preferred are the primer pairs of Figure A(2) and Figure B(2).
（２）本発明のプライマー対Ｗについて (2) Primer pair W of the present invention
　本発明のプライマー対Ｗの例としては、塩基配列の置換又は挿入変異を検出する際に用いられる野生型検出用プライマー対（Ｗ－１）、又は塩基の欠損変異を検出する際に用いられる野生型検出用プライマー対（Ｗ－２）が挙げられる。 Examples of the primer pair W of the present invention include a wild-type detection primer pair (W-1) used for detecting substitution or insertion mutation of a base sequence, or a wild-type detection primer pair (W-1) used for detecting base deletion mutation. Type-detecting primer pair (W-2) can be mentioned.
１）本発明のプライマー対（Ｗ－１）
　塩基配列の置換又は挿入変異を検出する際に用いられる野生型検出用プライマー対である本発明のプライマー対（Ｗ－１）は、以下のものである。 1) Primer pair (W-1) of the present invention
The primer pair (W-1) of the present invention, which is a pair of primers for wild-type detection used for detecting substitution or insertion mutation in a nucleotide sequence, is as follows.
　野生型の二本鎖核酸の第２鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の置換又は挿入変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記野生型の二本鎖核酸の第１鎖（野生型の第１鎖）にアニールするプライマーであり、上記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の置換又は挿入変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 A primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand), and performs substitution or insertion mutation of the second strand of the mutant double-stranded nucleic acid (mutant-type second strand) Annealing to the region containing the nucleotide sequence in the wild-type second strand corresponding to the 3' complementary sequence of the nucleotide sequence of the wild-type second strand corresponding to the mutation in the mutant second strand The rest of the nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide sequence corresponding to the mutation of the mutant second strand. an F primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed;
A primer that anneals to the first strand of the wild-type double-stranded nucleic acid (wild-type first strand), and replaces the first strand of the mutant double-stranded nucleic acid (mutant first strand) or Annealing to the region containing the nucleotide sequence in the wild-type first strand corresponding to the insertion mutation, and the complementary sequence of the nucleotide sequence of the wild-type first strand corresponding to the mutation in the mutant first strand The other nucleotide sequence is the same as the complementary sequence of the 3'-side nucleotide sequence adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation of the mutant first strand. and a pair of R primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted.
　また、検出対象の塩基配列の置換又は挿入変異は、１～４塩基の置換又は挿入が好ましい。１～３塩基の置換又は挿入が好ましい。１塩基の置換又は挿入が更に好ましい。 Also, the substitution or insertion mutation of the base sequence to be detected is preferably a substitution or insertion of 1 to 4 bases. Substitutions or insertions of 1-3 bases are preferred. Substitution or insertion of a single base is more preferred.
　本発明のプライマー対Ｗ－１としては、以下のものが好ましい。 The following are preferred as the primer pair W-1 of the present invention.
　野生型の二本鎖核酸の第２鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の置換又は挿入変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、３’末端ヌクレオチドを含む連続する１～４個のヌクレオチドは上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列の相補配列と同じであって、その他の塩基配列は上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記野生型の二本鎖核酸の第１鎖（野生型の第１鎖）にアニールするプライマーであり、上記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の置換又は挿入変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、３'末端ヌクレオチドを含む連続する１～４個のヌクレオチドは上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列の相補配列と同じであって、その他の塩基配列は上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 A primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand), and performs substitution or insertion mutation of the second strand of the mutant double-stranded nucleic acid (mutant-type second strand) Anneals to a region containing a base sequence in the wild-type second strand corresponding to the above, and 1 to 4 consecutive nucleotides including the 3′ terminal nucleotide correspond to the mutation of the mutant second strand 3, which is the same as the complementary sequence of the base sequence of the wild-type second strand, and has other base sequences adjacent to the base sequence of the wild-type second strand corresponding to the mutation of the mutant-type second strand; It is the same as the complementary sequence of the base sequence on the 'side, and the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-ified. and an F primer
A primer that anneals to the first strand of the wild-type double-stranded nucleic acid (wild-type first strand), and replaces the first strand of the mutant double-stranded nucleic acid (mutant first strand) or Annealed to a region containing a nucleotide sequence in the wild-type first strand corresponding to the insertion mutation, and 1 to 4 consecutive nucleotides including the 3′ terminal nucleotide correspond to the mutation in the mutant first strand and the other nucleotide sequence is adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation in the mutant first strand. is the same as the complementary sequence of the base sequence on the 3' side, and the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S A primer pair for the R primer that is labeled.
　本発明のプライマー対Ｗ－１としては、以下のものがより好ましい。
　野生型の二本鎖核酸の第２鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の一塩基の置換又は挿入変異に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、３’末端ヌクレオチドは上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基の相補塩基と同じであって、その他の塩基配列は上記変異型の第２鎖の上記変異に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　上記野生型の二本鎖核酸の第１鎖（野生型の第１鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の一塩基の置換又は挿入変異に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、３'末端ヌクレオチドは上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基の相補塩基と同じであって、その他の塩基配列は上記変異型の第１鎖の上記変異に対応する上記野生型の第１鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーの、プライマー対。 As the primer pair W-1 of the present invention, the following are more preferable.
A primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand) and substitutes a single base in the second strand of a mutant double-stranded nucleic acid (mutant-type second strand) Or anneal to a region containing a nucleotide sequence in the wild-type second strand corresponding to the insertion mutation, and the 3' terminal nucleotide of the wild-type second strand corresponding to the mutation of the mutant second strand It is the same as the complementary base of the base, and the other base sequence is a complementary sequence of the base sequence on the 3' side adjacent to the base sequence of the wild-type second strand corresponding to the mutation in the second strand of the mutant type. and the F primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-formed,
A primer that anneals to the first strand of the wild-type double-stranded nucleic acid (wild-type first strand), the first strand of the mutant double-stranded nucleic acid (mutant-type first strand) The wild-type first strand that anneals to the region containing the nucleotide sequence in the wild-type first strand corresponding to the substitution or insertion mutation, and the 3′ terminal nucleotide corresponds to the mutation in the mutant-type first strand and the other base sequence is complementary to the base sequence on the 3' side adjacent to the base sequence of the wild-type first strand corresponding to the mutation of the first strand of the mutant type An R primer having the same sequence as the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-ified. versus.
　詳細は、後記する図Ａ２及び図Ｂ２を用いて説明する。 Details will be explained using Figures A2 and B2 below.
（ｉ）置換検出に用いられる野生型検出用プライマー対（Ｗ－１）について (i) Wild-type detection primer pair (W-1) used for substitution detection
　塩基配列の置換を検出する際に用いられる野生型検出用プライマー対である、本発明のプライマー対（Ｗ－１）について、下記図Ａ２をもとに説明する。
　図Ａ２では、各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 The primer pair (W-1) of the present invention, which is a wild-type detection primer pair used for detecting substitution of base sequences, will be described with reference to FIG. A2 below.
FIG. A2 exemplifies the case where the number of S-nucleotides in each primer is three.
[図Ａ２]
Figure JPOXMLDOC01-appb-I000010
[Figure A2]
Figure JPOXMLDOC01-appb-I000010
図Ａ２(１)：１塩基置換検出用プライマー対（野生型）
　図Ａ２(１)は、１塩基置換を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ａ２(１)では、野生型の塩基配列に対し●印の塩基が、第１鎖ではgがaに置換され、第２鎖ではcがtに置換されている１塩基置換を検出対象とする場合を示す。
　Ｆプライマーは、１塩基置換を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異塩基(t)に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基であるcの相補塩基(g)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記変異塩基(t)に対応する上記野生型の第２鎖の塩基であるcに隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、上記１塩基置換を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異塩基(a)に対応する野生型二本鎖核酸の第１鎖（野生型の第１鎖）の塩基であるgの相補塩基(c)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記変異塩基(a)に対応する上記野生型の第１鎖の塩基であるgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A2 (1): Primer pair for detecting single base substitution (wild type)
FIG. A2(1) shows an example of the primer pair (W-1) of the present invention used for wild type detection when detecting a single nucleotide substitution. In Fig. A2 (1), the detection target is a single base substitution in which the wild-type base sequence is replaced with a by g in the first strand, and c is replaced by t in the second strand. indicates when
The F primer is the second strand of a wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (g) of c, which is the base of the second strand of the type. The other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to c, which is the nucleotide of the wild-type second strand corresponding to the mutated nucleotide (t) of the second strand of the mutant type. Same as array (tgtgattggt). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of the wild-type double-stranded nucleic acid (wild It has at its 3' end the complementary base (c) of g, which is the base of the first strand of the type). The other nucleotide sequence is complementary to the nucleotide sequence (catccgc) on the 3′ side adjacent to g, which is the base of the wild-type first strand corresponding to the mutated nucleotide (a) of the first strand of the mutant type. Same as array (gcggatg). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
図Ａ２(２)：２塩基置換検出用プライマー対（野生型）
　図Ａ２(２)は、２塩基置換を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ａ２では、野生型の塩基配列に対し、●印の塩基が、第１鎖ではtgがcaに置換され、第２鎖ではcaがtgに置換されている２塩基置換を検出対象とする場合を示す。
　Ｆプライマーは、２塩基置換を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異塩基配列(tg)に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基配列であるcaの相補配列(tg)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記変異塩基配列（tg）に対応する上記野生型の第２鎖の塩基配列であるcaに隣接する３’側の塩基配列（ccaatcaca）の相補配列(tgtgattgg)と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、上記２塩基置換を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異塩基配列(ca)に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基配列であるtgの相補配列(ca)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記変異塩基配列(ca)に対応する上記野生型の第１鎖の塩基配列であるtgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（cgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A2 (2): Primer pair for detecting double base substitution (wild type)
FIG. A2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting a two-base substitution. In FIG. A2, for the base sequence of the wild-type, when the bases marked with ● are to be detected, tg is substituted with ca in the first strand, and ca is substituted with tg in the second strand. indicates
The F primer is the second strand of a wild-type double-stranded nucleic acid ( It has a complementary sequence (tg) of ca, which is the nucleotide sequence of the wild-type second strand), at its 3' end. And the other nucleotide sequence is the 3′-side nucleotide sequence (ccaatcaca) adjacent to ca, which is the nucleotide sequence of the wild-type second strand corresponding to the mutant nucleotide sequence (tg) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattgg) of In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated base sequence (ca) of the first strand of the mutant double-stranded nucleic acid having the above two base substitutions (mutant first strand). It has a complementary sequence (ca) of tg, which is the base sequence of (wild-type first strand), at its 3' end. The other base sequence is the base sequence (catccgc) on the 3' side adjacent to tg, which is the base sequence of the wild-type first strand corresponding to the mutant base sequence (ca) of the first strand of the mutant type. is the same as the complementary sequence (gcggatg) of In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the R primer toward the 5' side (cgt) is S-formed. .
図Ａ２(３)：３塩基置換検出用プライマー対（野生型）
　図Ａ２(３)は、３塩基が置換された変異を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ａ２(３)では、野生型の塩基配列に対し、●印の塩基が、第１鎖ではgtgがtcaに置換され、第２鎖ではcacがtgaに置換されている３塩基置換を検出対象とする場合を示す。
　Ｆプライマーは、３塩基置換を有する変異型の二本鎖核酸の第２鎖（野生型の第２鎖）の変異塩基配列(tga)に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基配列であるcacの相補配列(gtg)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記変異塩基配列(tga)に対応する上記野生型の第２鎖の塩基配列であるcacに隣接する３’側の塩基配列（caatcaca）の相補配列（tgtgattg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、上記３塩基置換を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異塩基配列(tca)に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基配列であるgtgの相補配列(cac)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記変異塩基配列（tca）に対応する上記野生型の第１鎖の塩基配列であるgtgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（acg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure A2 (3): Primer pair for detecting 3 base substitutions (wild type)
FIG. A2(3) shows an example of the primer pair (W-1) of the present invention, which is used for wild-type detection when detecting mutations in which three bases are substituted. In Fig. A2 (3), for the base sequence of the wild type, detection target is 3 base substitutions in which gtg is substituted with tca in the first strand and cac is substituted with tga in the second strand. A case where
The F primer is a wild-type double-stranded nucleic acid second strand ( It has a complementary sequence (gtg) of cac, which is the nucleotide sequence of the wild-type second strand), at its 3' end. The other base sequence is the base sequence (caatcaca) on the 3' side adjacent to cac, which is the base sequence of the wild-type second strand corresponding to the mutant base sequence (tga) of the second strand of the mutant type. is the same as the complementary sequence (tgtgattg) of In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid corresponding to the mutated nucleotide sequence (tca) of the first strand of the mutant double-stranded nucleic acid having the three base substitutions (mutant first strand). It has a gtg complementary sequence (cac), which is the base sequence of (wild-type first strand), at its 3' end. The other base sequence is the base sequence (catccgc) on the 3' side adjacent to the base sequence gtg of the wild-type first strand corresponding to the mutant base sequence (tca) of the first strand of the mutant type. is the same as the complementary sequence (gcggatg) of In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (acg) from the second base from the 3' end of the R primer toward the 5' side (acg) is S-formed. .
(ii)挿入検出に用いられる野生型検出用プライマー対（Ｗ－１）について
　塩基配列の挿入を検出する際に用いられる野生型検出用プライマー対である、本発明のプライマー対（Ｗ－１）について、下記図Ｂ２をもとに説明する。
　図Ｂ２では、各プライマーのＳ化されたヌクレオチドの数が３個の場合を例示する。 (ii) Wild-type detection primer pair (W-1) used for insertion detection The primer pair (W-1) of the present invention, which is a wild-type detection primer pair used for detecting insertion of a nucleotide sequence will be explained based on the following diagram B2.
FIG. B2 exemplifies the case where the number of S-nucleotides in each primer is three.
[図Ｂ２]
Figure JPOXMLDOC01-appb-I000011
[Figure B2]
Figure JPOXMLDOC01-appb-I000011
図Ｂ２(１)：１塩基挿入検出用プライマー対（野生型）
　図Ｂ２(１)は、１塩基挿入を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ｂ２(１)では、野生型の塩基配列に対し、矢印の位置に、第１鎖ではtが挿入され、第２鎖ではaが挿入された１塩基挿入を検出対象とする場合を示す。
　Ｆプライマーは、１塩基挿入を有する変異型二本鎖核酸の第２鎖（変異型の第２鎖）の挿入変異に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の位置に隣接する５’側の塩基(g)の相補塩基であるcを、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記挿入変異に対応する野生型の第２鎖の位置に隣接する３’側の塩基配列（caccaatcaca）の相補配列（tgtgattggtg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gtg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、上記１塩基挿入を有する変異型二本鎖核酸の第１鎖（変異型の第１鎖）の挿入変異に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の位置に隣接する３’側の塩基（c）の相補塩基であるgを、その３’末端に有する。そしてその他の塩基配列は、上記（変異型の第１鎖の上記挿入変異に対応する野生型の第１鎖の位置に隣接する）上記３’側の塩基（c）に隣接する３’側の塩基配列（atccgc）の相補配列（cgcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B2 (1): Primer pair for detecting single base insertion (wild type)
FIG. B2(1) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when single-base insertion is detected. FIG. B2(1) shows the case of detecting a single base insertion in which t is inserted in the first strand and a is inserted in the second strand at the positions indicated by the arrows in the wild-type base sequence.
The F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end c, the complementary base of the 5' base (g) adjacent to the (strand) position. The other nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the 3′-side nucleotide sequence (caccaatcaca) adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand. be. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
The R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. and the other base sequence is the 3′-side base (c) adjacent to the 3′-side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the base sequence (atccgc). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
図Ｂ２(２)：２塩基挿入検出用プライマー対（野生型）―１
　図Ｂ２(２)は、２塩基挿入を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ｂ２(２)では、野生型の塩基配列に対し、矢印の位置に第１鎖ではtaが挿入され、第２鎖ではtaが挿入された２塩基挿入を検出対象とする場合を示す。
　Ｆプライマーは、２塩基挿入を有する変異型二本鎖核酸の第２鎖（変異型の第２鎖）の挿入変異に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の位置を挟む塩基配列（gc）の相補配列であるgcを、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記挿入変異に対応する野生型の第２鎖の位置を挟む塩基配列（gc）に隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gtg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、２塩基挿入を有する変異型二本鎖核酸の第１鎖（変異型の第１鎖）の挿入変異に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の位置を挟む塩基配列（gc）の相補配列であるgcを、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記挿入変異に対応する野生型の第１鎖の位置を挟む塩基配列（gc）に隣接する３’側の塩基配列（atccgc）の相補配列（cggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（ggt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B2 (2): Primer pair for detecting double base insertion (wild type)-1
FIG. B2(2) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion. FIG. B2(2) shows a case in which 2-base insertions in which ta is inserted in the first strand and ta is inserted in the second strand at the positions indicated by arrows in the wild-type base sequence are detected.
The F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand). The other nucleotide sequence is complementary to the nucleotide sequence (accaatcaca) on the 3′ side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type second strand corresponding to the insertion mutation of the second strand of the mutant type. Same as array (tgtgattggt). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) extending from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end gc, which is the complementary sequence of the base sequence (gc) flanking the position of the strand). The other nucleotide sequence is the complement of the nucleotide sequence (atccgc) on the 3' side adjacent to the nucleotide sequence (gc) flanking the position of the wild-type first strand corresponding to the insertion mutation of the first strand of the mutant type. Same as array (cggatg). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (ggt) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ｂ２(３)：２塩基挿入検出用プライマー対（野生型）―２
　図Ｂ２(３)は、２塩基挿入を検出する際に、野生型の検出に用いられる本発明のプライマー対（Ｗ－１）の例を示す。図Ｂ２(３)では、野生型の塩基配列に対し、矢印の位置に第１鎖ではtaが挿入され、第２鎖ではtaが挿入された２塩基挿入を検出対象とする場合の、図Ｂ２(２)とは別の例を示す。
　Ｆプライマーは、２塩基挿入を有する変異型二本鎖核酸の第２鎖（変異型の第２鎖）の挿入変異に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の位置の５’側の塩基配列であるgtの相補配列であるcaを、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記挿入変異に対応する野生型の第２鎖の位置に隣接する３’側の塩基配列（caccaatcaca）の相補配列（tgtgattggtg）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるcから５’側に向けての連続する３個のヌクレオチド（cgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、上記１塩基挿入を有する変異型二本鎖核酸の第１鎖（変異型の第１鎖）の挿入変異に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の位置に隣接する３’側の塩基（c）の相補塩基であるgを、その３’末端に有する。そしてその他の塩基配列は、上記（変異型の第１鎖の上記挿入変異に対応する野生型の第１鎖の位置に隣接する）上記３’側の塩基（c）に隣接する３’側の塩基配列（atccgc）の相補配列（cgcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure B2 (3): Primer pair for detecting double base insertion (wild type)-2
FIG. B2(3) shows an example of the primer pair (W-1) of the present invention used for wild-type detection when detecting double base insertion. In FIG. B2(3), in the wild-type base sequence, ta is inserted in the first strand at the position of the arrow, and 2 base insertions in which ta is inserted in the second strand are to be detected. An example different from (2) is shown.
The F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end ca, which is a complementary sequence of gt, which is the 5' base sequence of the (strand) position. The rest of the nucleotide sequence is the same as the complementary sequence (tgtgattggtg) of the nucleotide sequence (caccaatcaca) on the 3' side adjacent to the position of the wild-type second strand corresponding to the insertion mutation of the mutant second strand. be. In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the F primer toward the 5' side (cgt) is S-formed. .
The R primer is the first strand of the wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the insertion mutation of the first strand of the mutant double-stranded nucleic acid having the single-base insertion (mutant first strand). It has at its 3' end g, the complementary base of the 3' base (c) adjacent to the 1 strand) position. And other nucleotide sequences are the 3′ side adjacent to the 3′ side base (c) (adjacent to the position of the wild-type first strand corresponding to the insertion mutation of the mutant first strand). It is the same as the complementary sequence (cgcggatg) of the nucleotide sequence (atccgc). In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
　本発明のプライマー対Ｗ－１の具体例として、例えばα１アンチトリプシン遺伝子の１塩基置換変異であるPiS変異検出に用いられる野生型検出用プライマー対（本発明のプライマー対Ｗ－１、上記図Ａ２（２）の場合に相当する。）を例にとり、以下に説明する。 As a specific example of the primer pair W-1 of the present invention, for example, a wild-type detection primer pair (primer pair W-1 of the present invention, FIG. A2 This corresponds to the case of (2).) will be described below as an example.
　α１アンチトリプシンの遺伝子変異であるPiS変異の詳細は上記した通りである The details of the PiS mutation, which is a gene mutation of α1-antitrypsin, are as described above.
　この１塩基置換の検出に用いられる野生型検出用プライマー対である本発明のプライマー対Ｗ－１を設計するにあたり、α１アンチトリプシンの遺伝子の二本鎖DNAの、AがTに置換された方の鎖を第１鎖とする。上記二本鎖DNAの、同じ部分の塩基のTがAに置換された方の鎖を、第２鎖とする。
　尚、本明細書で記載する各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 In designing the primer pair W-1 of the present invention, which is a wild-type detection primer pair used for detecting this single base substitution, the double-stranded DNA of the α1-antitrypsin gene in which A was replaced with T was used. is the first strand. The strand of the above double-stranded DNA in which T in the same portion of the base is replaced with A is referred to as the second strand.
In addition, in the base sequences of the primers described herein, the * mark indicates the position where the phosphodiester bond is S-converted.
PiS野生型検出用プライマー対（Ｗ－１）としては、例えば下記のものが設計される。 As the PiS wild-type detection primer pair (W-1), for example, the following are designed.
i)１塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCTGG*A-3’ （配列番号２７）
　Ｒプライマー: 5’-GTGATGATATCGTGGGTGAGTTCATTT*T-3’（配列番号２８） i) 1 base S primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCTGG*A-3' (SEQ ID NO: 27)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCATTT*T-3' (SEQ ID NO: 28)
ii)２塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCTG*G*A-3’ （配列番号３１）
　Ｒプライマー: 5’-GTGATGATATCGTGGGTGAGTTCATT*T*T-3’（配列番号３２）
iii)３塩基Ｓ化プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*A-3’（配列番号３５）
　Ｒプライマー: 5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3’（配列番号３６） ii) 2-base S-primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCTG*G*A-3' (SEQ ID NO: 31)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCATT*T*T-3' (SEQ ID NO: 32)
iii) 3-base S-primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*A-3' (SEQ ID NO: 35)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3' (SEQ ID NO: 36)
　PiS変異検出に用いられる野生型検出用プライマー対のＦプライマーは、PiS変異を有する二本鎖核酸の第２鎖（変異型の第２鎖）の変異した塩基(A)に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基(T)の相補塩基であるAを、３'末端に有する。その他の塩基配列は、上記変異型の第２鎖の変異塩基(A)に対応する上記野生型の第２鎖の塩基(T)に隣接する３’側の塩基配列の相補配列と同じになるように設計される。
　上記プライマー対のＲプライマーは、PiS変異を有する二本鎖核酸の第１鎖（変異型の第１鎖）の変異した塩基(T)に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基(A)の相補塩基であるTを、３'末端に有する。その他の塩基配列は、上記変異型の第１鎖の変異塩基(T)に対応する上記野生型の第１鎖の塩基(A)に隣接する３’側の塩基配列と同じになるように設計される。
　更に上記のプライマー対の例では３'末端から２～４番目のヌクレオチドは、そのホスホジエステル結合がＳ化されたヌクレオチドである。 The F primer of the wild-type detection primer pair used for PiS mutation detection is a wild-type corresponding to the mutated base (A) of the second strand of the double-stranded nucleic acid having a PiS mutation (second strand of the mutant type). It has A, which is the complementary base of the base (T) of the second strand (wild-type second strand) of the double-stranded nucleic acid, at the 3′ end. Other nucleotide sequences are the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type second strand nucleotide (T) corresponding to the mutant second strand nucleotide (A). is designed to
The R primer of the primer pair is the first strand of a wild-type double-stranded nucleic acid ( It has a T at the 3′ end, which is the complementary base of base (A) of wild-type first strand). Other base sequences are designed to be the same as the base sequence on the 3' side adjacent to the wild-type first-strand base (A) corresponding to the mutant first-strand base (T). be done.
Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3' end are nucleotides whose phosphodiester bonds are S-converted.
　PiS野生型検出用プライマー対の設計方法の一例を以下に説明する。
　Ｆプライマーは、その３'末端塩基はPiS遺伝子の変異塩基(T)に対応する野生型の塩基(A)と同じであり、その他の塩基配列は、PiS遺伝子の野生型の二本鎖核酸の第１鎖の上記変異塩基(T)に対応する野生型の塩基(A)より５’側に向けての塩基配列と同じになるように設計される。
　一方Ｒプライマーは、その３'末端塩基はPiS遺伝子の変異塩基(T)に対応する野生型の塩基(T)の相補塩基(A)と同じであり、その他の塩基配列は、PiS遺伝子の野生型の二本鎖核酸の第１鎖の上記変異塩基(T)に対応する野生型の塩基(A)に隣接する３’側に向けての塩基配列の相補配列と同じになるように設計される。
　更に上記のプライマー対の例では３'末端から２～４番目のヌクレオチドは、そのホスホジエステル結合がＳ化されたヌクレオチドとする。 An example of a method for designing a PiS wild-type detection primer pair is described below.
The F primer has the same 3′ terminal base as the wild-type base (A) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type double-stranded nucleic acid of the PiS gene. It is designed to have the same nucleotide sequence as the 5′ side of the wild-type nucleotide (A) corresponding to the above-mentioned mutated nucleotide (T) of the first strand.
On the other hand, the R primer has the same 3′ terminal base (A) as the complementary base (A) of the wild-type base (T) corresponding to the mutated base (T) of the PiS gene, and the other base sequence is the wild-type base (T) of the PiS gene. designed to be the same as the complementary sequence of the base sequence toward the 3′ side adjacent to the wild-type base (A) corresponding to the mutated base (T) of the first strand of the double-stranded nucleic acid of the type be.
Furthermore, in the example of the above primer pair, the 2nd to 4th nucleotides from the 3'-end are nucleotides whose phosphodiester bonds are S-converted.
　本発明のプライマー対Ｗのその他の例としては、例えばα１アンチトリプシンの別の遺伝子変異であるPiZ変異（１塩基変異）の検出に用いられる野生型検出用プライマー対が挙げられる。 Other examples of the primer pair W of the present invention include a wild-type detection primer pair used for detecting PiZ mutation (single nucleotide mutation), which is another gene mutation of α1-antitrypsin.
i)１塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCGAC*G-3’ （配列番号４１）
　Ｒプライマー:5’-CCCCAGCAGCTTCAGTCCCTTTCT*C-3’ （配列番号４２） i) 1 base S primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCGAC*G-3' (SEQ ID NO: 41)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTTCT*C-3' (SEQ ID NO: 42)
ii)２塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCGA*C*G-3’ （配列番号４５）
　Ｒプライマー:5’-CCCCAGCAGCTTCAGTCCCTTTC*T*C-3’ （配列番号４６） ii) 2-base S-primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCGA*C*G-3' (SEQ ID NO: 45)
R primer: 5'-CCCCAGCAGCTTTCAGTCCCTTTC*T*C-3' (SEQ ID NO: 46)
iii)３塩基Ｓ化プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCG*A*C*G-3’ （配列番号４９）
　Ｒプライマー:5’-CCCCAGCAGCTTCAGTCCCTTT*C*T*C-3’ （配列番号５０） iii) 3-base S-primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*G-3' (SEQ ID NO: 49)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*C-3' (SEQ ID NO: 50)
　本発明のプライマー対Ｗ－１のその他の例としては、例えば下記表３に記載のものが挙げられる。 Other examples of the primer pair W-1 of the present invention include those listed in Table 3 below.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
２）本発明のプライマー対（Ｗ－２）
　塩基配列の欠損を検出する際に用いられる野生型検出用プライマー対である本発明のプライマー対（Ｗ－２）は、以下のものである。
　野生型の二本鎖核酸の第２鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の欠損に対応する上記野生型の第２鎖中の塩基配列を含む領域にアニールし、上記変異型の第２鎖の欠損に対応する上記野生型の第２鎖の塩基配列の相補配列、又は上記野生型の第２鎖の塩基配列とそれに隣接する５’側の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第２鎖の上記欠損に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＦプライマーと、
　野生型の二本鎖核酸の第１鎖（野生型の第１鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第一鎖（変異型の第１鎖）の欠損に対応する上記野生型の第１鎖中の塩基配列を含む領域にアニールし、上記変異型の第１鎖の欠損に対応する上記野生型の第１鎖の塩基配列の相補配列、又は上記野生型の第１鎖の塩基配列とそれに隣接する５’側の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は上記変異型の第１鎖の上記欠損に対応する上記野生型の第２鎖の塩基配列に隣接する３’側の塩配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているＲプライマーのプライマー対。 2) Primer pair (W-2) of the present invention
The primer pair (W-2) of the present invention, which is a pair of primers for wild-type detection used for detecting deletion of nucleotide sequences, is as follows.
A primer that anneals to the second strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a defect in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) A complementary sequence of the base sequence of the wild-type second strand that anneals to the region containing the base sequence in the wild-type second strand and corresponds to the deletion of the mutant-type second strand, or the wild-type first strand It has a complementary sequence of the base sequence of the second strand and the base sequence of the 5' side adjacent to it at the 3' end side, and the other base sequence is the wild-type second strand corresponding to the deletion of the second strand of the mutant type. It is the same as the complementary sequence of the base sequence on the 3' side adjacent to the two-stranded base sequence, and is 3' of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side. an F primer in which the phosphodiester bond on the side is S-ified;
A primer that anneals to the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) and corresponds to a defect in the first strand of a mutant-type double-stranded nucleic acid (mutant-type first strand) A complementary sequence of the base sequence of the wild-type first strand that anneals to the region containing the base sequence in the wild-type first strand and corresponds to the deletion of the mutant-type first strand, or the wild-type first strand The first strand of the wild type has a sequence complementary to the base sequence of the first strand and the base sequence of the 5' side adjacent thereto at the 3' end side, and the other base sequence is the first strand of the wild type corresponding to the deletion of the first strand of the mutant type. It is the same as the complementary sequence of the 3' side salt sequence adjacent to the base sequence of the two strands, and 3' of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side A primer pair of R primers in which the flanking phosphodiester bonds are S ylated.
　詳細は、後記する図Ｃ２及び図Ｄ２を用いて説明する。 Details will be explained using Figures C2 and D2 below.
（ｉ）１塩基欠損検出に用いられる野生型検出用プライマー対（Ｗ－２）について
　塩基配列の１塩基欠損を検出する際に用いられる野生型検出用プライマー対である、本発明のプライマー対（Ｗ－２）について、下記図Ｃ２をもとに説明する。
　図Ｃ２では、野生型の塩基配列に対し、第１鎖では矢印のgが欠損しており、第２鎖では矢印のcが欠損している１塩基欠損を検出対象とする場合を示す。 (i) Wild-type detection primer pair (W-2) used for detecting 1-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 1-base deletion in a nucleotide sequence ( W-2) will be explained with reference to Figure C2 below.
FIG. C2 shows a case where a 1-base deletion is targeted for detection, in which the first strand lacks the arrow g and the second strand lacks the arrow c in the wild-type base sequence.
　図Ｃ２（１）～図Ｃ２（３）において、塩基配列の空欄は塩基が欠損していることを示し、実際には空欄の５'側の塩基と３’側の塩基は連続している。 In Figures C2(1) to C2(3), blanks in the base sequences indicate that bases are missing, and in fact, the 5'-side and 3'-side bases of the blanks are continuous.
　尚、図Ｃ２では各プライマーのＳ化されたヌクレオチドの数が３個の場合を示す。 Note that FIG. C2 shows the case where the number of S-nucleotides in each primer is 3.
[図Ｃ２]
Figure JPOXMLDOC01-appb-I000013
[Figure C2]
Figure JPOXMLDOC01-appb-I000013
図Ｃ２(１)：１塩基欠損検出用プライマー対（野生型）―１
　Ｆプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の上記欠損に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基であるcの相補塩基(g)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記欠損に対応する上記野生型の第２鎖の塩基であるcに隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の上記欠損に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基であるgの相補塩基(c)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記欠損に対応する上記野生型の第１鎖の塩基であるgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C2 (1): Primer pair for detecting 1 base deletion (wild type) -1
The F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand). The other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand). The other nucleotide sequence is a complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-converted. .
図Ｃ２(２)：１塩基欠損検出用プライマー対（野生型）―２
　Ｆプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の上記欠損に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基であるcの相補塩基(g)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記欠損に対応する上記野生型の第２鎖の塩基であるcに隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるtから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の上記欠損に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基であるgとそれに隣接する５’側の塩基(t)の、相補配列（ca）を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記欠損に対応する上記野生型の第２鎖の塩基であるgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatgc）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（cgt）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C2 (2): Primer pair for detecting 1 base deletion (wild type) -2
The F primer is the second strand of the wild-type double-stranded nucleic acid (wild-type second strand) corresponding to the deletion of the second strand of the mutant double-stranded nucleic acid having a single base deletion (mutant second strand). It has at its 3′ end the complementary base (g) of c, which is the base of the double strand). The other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3' end a complementary sequence (ca) of the base g of the first strand) and the adjacent 5' base (t). The other nucleotide sequence is a complementary sequence (gcggatgc) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the second strand of the wild type corresponding to the deletion of the first strand of the mutant type. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cgt) from the second base from the 3' end of the R primer toward the 5' side (cgt) is S-formed. .
図Ｃ２(３)：１塩基欠損検出用プライマー対（野生型）―３
　Ｆプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の欠損に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基であるcとそれに隣接する５’側の塩基(g)の、相補配列（gc）を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記欠損に対応する上記野生型の第２鎖の塩基であるcに隣接する３’側の塩基配列（accaatcaca）の相補配列（tgtgattggt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（gtg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、１塩基欠損を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の上記欠損に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基であるgの相補塩基(c)を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記欠損に対応する上記野生型の第１鎖の塩基であるgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるgから５’側に向けての連続する３個のヌクレオチド（gta）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure C2 (3): Primer pair for detecting 1 base deletion (wild type) -3
The F primer is the second strand of a wild-type double-stranded nucleic acid (wild-type second It has at its 3' end a complementary sequence (gc) of the base c of the chain) and the adjacent 5' base (g). The other nucleotide sequence is the complementary sequence (tgtgattggt) of the 3′-side nucleotide sequence (accaatcaca) adjacent to c, which is the base of the wild-type second strand corresponding to the deletion of the mutant-type second strand. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gtg) from the second base from the 3' end of the F primer toward the 5' side (gtg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first strand) corresponding to the deletion of the first strand of a mutant double-stranded nucleic acid having a single base deletion (mutant first strand). It has at its 3′ end the complementary base (c) of g, which is the base of the first strand). The other nucleotide sequence is the complementary sequence (gcggatg) of the 3′-side nucleotide sequence (catccgc) adjacent to g, which is the base of the wild-type first strand corresponding to the deletion of the mutant-type first strand. is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (gta) from the second base g from the 3' end of the R primer toward the 5' side is S-formed. .
（ii）４塩基欠損検出に用いられる野生型検出用プライマー対（Ｗ－２）について
　塩基配列の４塩基欠損を検出する際に用いられる野生型検出用プライマー対である、本発明のプライマー対（Ｗ－２）について、下記図Ｄ２をもとに説明する。
　図Ｄ２では、野生型の塩基配列に対し、第１鎖では●印のggtgが欠損しており、第２鎖では、●印のcaccが欠損している４塩基欠損を検出対象とする場合を示す。 (ii) Wild-type detection primer pair (W-2) used for detecting 4-base deletion The primer pair of the present invention, which is a wild-type detection primer pair used for detecting 4-base deletion in a nucleotide sequence ( W-2) will be explained based on the following diagram D2.
In FIG. D2, for the base sequence of the wild type, the first strand lacks ggtg marked with ●, and the second strand lacks cacc marked with ●. show.
　図Ｄ２において、塩基配列の空欄は塩基が欠損していることを示し、実際には空欄の５'側の塩基と３'側の塩基は連続している。
　尚、図Ｄ２では各プライマーのＳ化されたヌクレオチドの数が３個の場合を示す。 In FIG. D2, a blank in the base sequence indicates that a base is missing, and the 5'-side and 3'-side bases of the blank are actually continuous.
In addition, FIG. D2 shows the case where the number of S-nucleotides in each primer is three.
[図Ｄ２]
Figure JPOXMLDOC01-appb-I000014
[Figure D2]
Figure JPOXMLDOC01-appb-I000014
図Ｄ２：４塩基欠損検出用プライマー対
　Ｆプライマーは、４塩基欠損を有する変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の欠損に対応する野生型の二本鎖核酸の第２鎖（野生型の第２鎖）の塩基配列であるcaccの相補配列（ggtc）を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第２鎖の上記欠損に対応する上記野生型の第２鎖の塩基配列であるcaccに隣接する３’側の塩基配列（aatcaca）の相補配列（tgtgatt）と同じである。また、このＦプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（tgg）は、それぞれ３'側のホスホジエステル結合がＳ化されている。
　Ｒプライマーは、４塩基欠損を有する変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の欠損に対応する野生型の二本鎖核酸の第１鎖（野生型の第１鎖）の塩基配列であるggtgの相補配列（cacc）を、その３’末端に有する。そしてその他の塩基配列は、上記変異型の第１鎖の上記欠損に対応する上記野生型の第２鎖の塩基配列であるggtgに隣接する３’側の塩基配列（catccgc）の相補配列（gcggatg）と同じである。また、このＲプライマーの３’末端から２番目の塩基であるaから５’側に向けての連続する３個のヌクレオチド（cac）は、それぞれ３'側のホスホジエステル結合がＳ化されている。 Figure D2: Primer pair for detecting 4-base deletion The F primer is a wild-type double-stranded nucleic acid corresponding to the deletion of the second strand of a mutant double-stranded nucleic acid having a 4-base deletion (mutant second strand). It has at its 3′ end a complementary sequence (ggtc) of cacc, which is the base sequence of the second strand (wild-type second strand). The other nucleotide sequence is a complementary sequence (tgtgatt ) is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (tgg) from the second base from the 3' end of the F primer toward the 5' side (tgg) is S-formed. .
The R primer is the first strand of a wild-type double-stranded nucleic acid (wild-type first It has at its 3' end the complementary sequence (cacc) of ggtg, which is the base sequence of the ggtg chain). The other nucleotide sequence is a complementary sequence (gcggatg ) is the same as In addition, the phosphodiester bond on the 3' side of each of the three consecutive nucleotides (cac) from the second base from the 3' end of the R primer to the 5' side (cac) is S-formed. .
　尚、特に図を用いて説明はしないが、２塩基欠損又は３塩基欠損を検出するプライマー対Ｗ－２も、同様の方法で設計することができる。 A primer pair W-2 for detecting a 2-base deletion or a 3-base deletion can also be designed by a similar method, although not explained using the figures.
　本発明のプライマー対Ｗ－２の具体例としては、例えば下記のものが挙げられる。 Specific examples of the primer pair W-2 of the present invention include the following.
　例えば、上記したイヌのVPS13B遺伝子の４塩基欠損を検出するための野生型検出用プライマー対として、下記のプライマー対が設計される。 For example, the following primer pair is designed as a wild-type detection primer pair for detecting the above-described 4-base deletion in the canine VPS13B gene.
・VPS13B野生型野生型検出用プライマー対
　Ｆプライマー：5’-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T*T-3’（配列番号７１）
　Ｒプライマー: 5’-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3’（配列番号７２） - VPS13B wild-type wild-type detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T*T-3' (SEQ ID NO: 71)
R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3' (SEQ ID NO: 72)
　本発明のプライマー対Ｗとしては、図Ａ２(２)、図Ｂ２(２)、図Ｃ２(２)、及び図Ｄ２(２)のプライマー対が好ましい。塩基配列変異の判定の容易さを考慮すると、図Ａ２(２)及び図Ｂ２(２)のプライマー対がより好ましい。 As the primer pair W of the present invention, the primer pairs shown in Figure A2(2), Figure B2(2), Figure C2(2), and Figure D2(2) are preferable. Considering the ease of determination of nucleotide sequence mutations, the primer pairs of Figure A2(2) and Figure B2(2) are more preferable.
　本発明のプライマー対Ｗ－２のその他の例としては、例えば下記表４に記載のものが挙げられる。 Other examples of the primer pair W-2 of the present invention include those listed in Table 4 below.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　上記表４において、塩基配列の下線を引いた塩基は、変異型において欠損する対象の塩基を示す。 In Table 4 above, the underlined bases in the base sequence indicate the bases to be deleted in the mutant.
＜２．本発明の塩基配列の変異の判定方法＞
　本発明の塩基配列の変異の判定方法は、
「被検試料の核酸を鋳型として用い、本発明のプライマー対（例えばプライマー対Ｍ又は／及びプライマー対Ｗ）を用いた核酸増幅反応を行って反応産物を検出し、得られた検出結果をもとに、上記核酸の塩基配列の変異を判定する、塩基配列の変異の判定方法。」
である。 <2. Method for Determining Nucleotide Sequence Mutation of the Present Invention>
The method for determining a nucleotide sequence mutation of the present invention comprises
"Using the nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair of the present invention (for example, primer pair M or/and primer pair W) to detect the reaction product, and the resulting detection result is also reported. and a method for determining a mutation in a base sequence, which determines a mutation in the base sequence of the nucleic acid."
is.
（１）被検試料
　本発明の塩基配列の変異の判定方法に用いられる被検試料としては、検体から採取された口腔拭い液（口腔スワブ）、鼻スワブ、鼻咽頭スワブ、咽頭スワブ、唾液、腹水，胸水，神経根周囲液，リンパ液，髄液，消化液等の体液、気管内吸引液、咽頭粘液、喀痰、経気管支採取物、気管支洗浄液、胸水等の各種臨床材料、血漿，血清，全血等の血液試料、組織切片、組織の洗浄液、糞便，尿等の生体由来試料、培養細胞、細胞上清、細胞溶解物、あるいはこれら試料に加熱処理等の前処理を行ったもの、これら試料から必要に応じて希釈若しくは濃縮等の調整を行ったもの、等が挙げられる。 (1) Test sample The test sample used in the method for determining a nucleotide sequence mutation of the present invention includes oral swabs (oral swabs), nasal swabs, nasopharyngeal swabs, pharyngeal swabs, saliva, Body fluids such as ascites, pleural effusion, nerve root fluid, lymphatic fluid, cerebrospinal fluid, digestive fluid, intratracheal aspirate, pharyngeal mucus, sputum, transbronchial sample, bronchial lavage, various clinical materials such as pleural effusion, plasma, serum, total Blood samples such as blood, tissue sections, tissue lavage fluids, feces, urine and other biological samples, cultured cells, cell supernatants, cell lysates, or pretreated samples such as heat-treated samples and those adjusted by dilution or concentration, etc., as necessary.
　本発明に係る被検試料が得られる検体としては、検出対象の遺伝子の核酸を含むものであれば、動物、植物、ウイルスその他、何等限定されない。 The specimen from which the test sample according to the present invention is obtained is not limited to animals, plants, viruses, etc., as long as it contains the nucleic acid of the gene to be detected.
　本発明に係る被検試料が得られる動物の例としては、哺乳類（ヒト、サル、ウシ、ブタ、ウマ、イヌ、ネコ、ヒツジ、ヤギ、ウサギ、ハムスター、モルモット、コウモリ、マウス、ラット等）、爬虫類等が挙げられる。 Examples of animals from which the test sample according to the present invention can be obtained include mammals (humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, bats, mice, rats, etc.), Examples include reptiles and the like.
　本発明に係る被検試料が得られるウイルスとしては、SARS-CoV-2等が挙げられる。 Viruses from which test samples according to the present invention can be obtained include SARS-CoV-2 and the like.
　本発明に係る遺伝子変異の検出に用いられる「被検試料の核酸」又は「被検試料由来の核酸」としては、上記した被検試料から抽出、精製された核酸、又は核酸増幅検出系等で増幅された核酸でもよい。また核酸は二本鎖でも一本鎖でもよく、DNAでもRNAでもよい。RNAの場合、逆転写反応を行って得られたcDNAを核酸として用いてもよい。 As the "test sample nucleic acid" or "test sample-derived nucleic acid" used for detecting gene mutation according to the present invention, a nucleic acid extracted and purified from the above-described test sample, or a nucleic acid amplification detection system, etc. It may be an amplified nucleic acid. Nucleic acids may be double-stranded or single-stranded, and may be DNA or RNA. In the case of RNA, cDNA obtained by reverse transcription may be used as the nucleic acid.
　本発明の塩基配列の判定方法に用いられる被検試料の核酸は、上記被検試料から、この分野で通常行われる方法で単離、精製すればよい。 The nucleic acid of the test sample used in the method for determining the nucleotide sequence of the present invention may be isolated and purified from the test sample by methods commonly used in this field.
（２）核酸増幅反応
　本発明の塩基配列の変異の判定方法に用いられるプライマー及びプライマー対については「＜１．本発明のプライマー対＞」の項で説明した通りであり、好ましい例、具体例等も同じである。 (2) Nucleic acid amplification reaction The primers and primer pairs used in the method for determining nucleotide sequence mutations of the present invention are as described in the section "<1. Primer pairs of the present invention>", and preferred examples and specific examples. etc. is the same.
　本発明の遺伝子変異の判定方法に係る核酸増幅反応は、本発明のプライマー対を用いる以外は、この分野で通常行われている核酸増幅反応を行えばよい。例えば、本発明のプライマー対を用いて、これを試料中の核酸とハイブリダイゼーションさせ、DNAポリメラーゼ等による核酸増幅[例えばPCR（polymerase chain reaction）、ＬＡＭＰ（Loop-mediated Isothermal Amplification）法、ICAN（Isothermal and Chimeric primer-initiated Amplification of Nucleic acids）法、LCR(ligase chain reaction)法、SDA(strand displacement amplification)法]を行ってプライマー伸長させる、常法が挙げられる。核酸増幅反応の条件、操作法等は、この分野で通常行われている常法に従えばよい。 For the nucleic acid amplification reaction according to the method for determining gene mutation of the present invention, a nucleic acid amplification reaction commonly performed in this field may be performed, except that the primer pair of the present invention is used. For example, using the primer pair of the present invention, it is hybridized with the nucleic acid in the sample, and nucleic acid amplification by DNA polymerase [for example, PCR (polymerase chain reaction), LAMP (Loop-mediated Isothermal Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, LCR (ligase chain reaction) method, SDA (strand displacement amplification) method] to extend the primer. The conditions, operation method, etc. of the nucleic acid amplification reaction may be in accordance with conventional methods commonly practiced in this field.
　本発明に係る「核酸増幅反応で得られた反応産物を検出する方法」としては、例えばインターカレーター法、TaqManTMリアルタイムPCR法、MGB Eclipse Probe System法、Molecular Beacons Probe Technology法、LUX Fluorogenic Primer法、Quenching probe-PCR（QP）法、核酸増幅反応を行った後、得られたプライマー伸長産物について電気泳動を行い、その結果に基づいて行う方法、標識プライマーを用いた核酸増幅反応を行って得られたプライマー伸長産物の標識を測定する方法等、様々な検出法が挙げられる。 The "method for detecting a reaction product obtained by a nucleic acid amplification reaction" according to the present invention includes, for example, the intercalator method, the TaqMan TM real-time PCR method, the MGB Eclipse Probe System method, the Molecular Beacons Probe Technology method, the LUX Fluorogenic Primer method, Quenching probe-PCR (QP) method, a method based on electrophoresis of the obtained primer extension product after performing nucleic acid amplification reaction, a method based on the result, a method obtained by performing nucleic acid amplification reaction using labeled primers A variety of detection methods are included, including methods that measure labeling of primer extension products.
　例えば本発明のプライマー対を用い、公知のインターカレーター（例えばEvaGreenTM（Biotium社製）、SYBRTM Green I (Molecular Probe社製）等)を利用して通常のインターカレーター法を利用したリアルタイムPCRを行いインターカレーターに由来する蛍光強度を検出する。 For example, using the primer pair of the present invention, real-time PCR using a known intercalator (e.g., EvaGreen ™ (manufactured by Biotium), SYBR ™ Green I (manufactured by Molecular Probe), etc.) is performed using a conventional intercalator method. Fluorescence intensity derived from the intercalator is detected.
　尚、被検試料由来の核酸が少量である場合には、通常行われている方法であるが、精製した被検試料由来の核酸を鋳型として核酸増幅反応を行い、上記核酸の検出対象の変異を含む領域を増幅させる（1st PCR）。そしてその増幅産物を鋳型として用い、本発明のプライマー対を用いた核酸増幅反応を行ってもよい。 When the amount of the nucleic acid derived from the test sample is small, a nucleic acid amplification reaction is performed using the purified nucleic acid derived from the test sample as a template, and the mutation to be detected in the nucleic acid is used. Amplify the region containing (1st PCR). Then, using the amplified product as a template, a nucleic acid amplification reaction using the primer pair of the present invention may be performed.
（３）変異を判定する方法
　変異を判定する方法としては、
　１）被検試料の核酸の塩基配列が野生型、ホモ接合型変異、又はヘテロ接合型変異かを判定する方法、又は
　２）被検試料の核酸の塩基配列が野生型か変異型かを判定する方法、
が挙げられる。
　以下に、それぞれの判定方法について説明する。 (3) Method for determining mutation As a method for determining mutation,
1) A method for determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation; or 2) Determining whether the nucleotide sequence of a nucleic acid in a test sample is wild-type or mutant. how to,
is mentioned.
Each determination method will be described below.
　１）被検試料の核酸の塩基配列が野生型、ホモ接合型変異、又はヘテロ接合型変異かを判定する方法について
　この判定方法としては、
『本発明のプライマー対Ｍ及び本発明のプライマー対Ｗを用い、下記の工程を含む判定方法：
　（i）被検試料の核酸を鋳型として用い、本発明のプライマー対Ｍを用いた核酸増幅反応を行い、反応産物を検出する工程、
　（ii）上記(i)の工程で用いたものと同じ被検試料の核酸を鋳型として用い、本発明のプライマー対Ｗを用いた核酸増幅反応を行い、反応産物を検出する工程、
　（iii）上記(i)及び(ii)の工程で得られた検出結果をもとに、被検試料の核酸の塩基配列の変異を判定する工程。』が挙げられる。 1) Method for determining whether the nucleotide sequence of the nucleic acid in the test sample is wild type, homozygous mutation, or heterozygous mutation As this determination method,
"Using the primer pair M of the present invention and the primer pair W of the present invention, a determination method comprising the following steps:
(i) using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M of the present invention, and detecting the reaction product;
(ii) a step of performing a nucleic acid amplification reaction using the primer pair W of the present invention using the nucleic acid of the same test sample as that used in step (i) above as a template, and detecting the reaction product;
(iii) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the above steps (i) and (ii). ] is mentioned.
　本発明のプライマー対Ｍ及び本発明のプライマー対Ｗについては「＜１．本発明のプライマー対＞」の項で説明した通りであり、好ましい例、具体例等も同じである。また、上記工程(i)及び工程(ii)における各プライマー対を用いた核酸増幅反応を行う方法は、「(２)核酸増幅反応」の項で説明した方法に準じてなされればよく、好ましい例、具体例等も同じである。
　尚、工程(i)と工程(ii)はどちらを先に行ってもよく、順序は問われない。 Primer pair M of the present invention and primer pair W of the present invention are as described in the section "<1. Primer pair of the present invention>", and preferred examples and specific examples are also the same. In addition, the method of carrying out the nucleic acid amplification reaction using each primer pair in the above steps (i) and (ii) may be carried out according to the method described in the section "(2) Nucleic acid amplification reaction", which is preferable. Examples, specific examples, etc. are the same.
Either step (i) or step (ii) may be performed first, and the order of the steps does not matter.
　工程(iii)は、工程(i)及び工程(ii)を行ってそれぞれ増幅曲線を取得し、その増幅曲線を、後記する方法で得られた対照の増幅曲線と比較することにより実施してもよい。 Step (iii) may be performed by performing steps (i) and (ii) to obtain an amplification curve, respectively, and comparing the amplification curve with a control amplification curve obtained by the method described below. good.
１－ａ）増幅曲線をもとに判定する方法
　被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かを判定する（すなわち遺伝子型を判定する）具体的な方法の一つとして、核酸増幅反応で測定した蛍光強度の増幅曲線をもとに判定を行う方法が挙げられる。以下に、被検試料由来の核酸を鋳型として用いて得られた増幅曲線を、対照の増幅曲線と比較して判定する方法を例にとって説明する。 1-a) Determining method based on amplification curve Determining whether the nucleotide sequence of the nucleic acid of the test sample is a wild type, homozygous mutation, or heterozygous mutation (that is, determining genotype) As one method, there is a method of making determination based on an amplification curve of fluorescence intensity measured in a nucleic acid amplification reaction. A method for judging by comparing an amplification curve obtained using a test sample-derived nucleic acid as a template with a control amplification curve will be described below as an example.
　１－ａ－１）対照の増幅曲線の作成
　検出対象の遺伝子の変異配列を含む、上記遺伝子の塩基配列（部分配列でよい）を有するオリゴヌクレオチド（変異型コントロール）、及び上記遺伝子の野生型の塩基配列を含み、上記変異に対応する塩基配列を含むオリゴヌクレオチド（野生型コントロール）を設計し、調製する。 1-a-1) Preparation of control amplification curve An oligonucleotide (mutant control) having the base sequence of the gene (may be a partial sequence) containing the mutation sequence of the gene to be detected, and the wild type of the gene An oligonucleotide (wild-type control) containing the nucleotide sequence corresponding to the above mutation is designed and prepared.
　また、上記＜１．本発明のプライマー対＞に記載の、検出対象の変異検出用プライマー対Ｍと、同じ検出対象の変異に対応する野生型検出用プライマー対Ｗを用い、下記表５の組合せのコントロールを鋳型として用い、本発明のプライマー対を用いて対照となる増幅曲線の見本を作成する。 Also, the above <1. Primer pairs of the present invention>, using a primer pair M for detecting a mutation to be detected and a primer pair W for wild-type detection corresponding to the same mutation to be detected, and using a control of the combination shown in Table 5 below as a template. , prepare a sample control amplification curve using the primer pairs of the invention.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　野生型コントロールを鋳型として用い、本発明のプライマー対Ｗを用いて核酸増幅反応を行い、増幅曲線を得る（増幅曲線１－１’）。また、同じ野生型コントロールを鋳型として用い、本発明のプライマー対Ｍを用いて同様に核酸増幅反応を行い、増幅曲線を得る（増幅曲線１－２’）。
　そして、得られた増幅曲線１－１’と増幅曲線１－２’を一つのグラフに表し、このグラフを野生型対照とする。 Using the wild-type control as a template, a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 1-1'). Further, using the same wild-type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 1-2').
Then, the resulting amplification curve 1-1' and amplification curve 1-2' are plotted in one graph, and this graph is used as a wild-type control.
　変異型コントロールを鋳型として用い、本発明のプライマー対Ｗを用いて核酸増幅反応を行い、増幅曲線を得る（増幅曲線２－１’）。また、同じ変異型コントロールを鋳型として用い、本発明のプライマー対Ｍを用いて同様に核酸増幅反応を行い、増幅曲線を得る（増幅曲線２－２’）。
　そして、得られた増幅曲線２－１’と増幅曲線２－２’を一つのグラフに表し、このグラフをヘテロ接合型対照とする。 Using the mutant control as a template, a nucleic acid amplification reaction is performed using the primer pair W of the present invention to obtain an amplification curve (amplification curve 2-1'). Further, using the same mutant type control as a template, a similar nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 2-2').
Then, the obtained amplification curve 2-1' and amplification curve 2-2' are represented in one graph, and this graph is used as a heterozygous control.
　上記と同じ野生型コントロールと上記と同じ変異型コントロールの混合物を鋳型として用い、本発明のプライマー対Ｗを用いて核酸増幅反応を行い、増幅曲線を得る（増幅曲線３－１’）。また、上記と同じ野生型コントロールと上記と同じ変異型コントロールの混合物を鋳型として用い、本発明のプライマー対Ｍを用いて同様に核酸増幅反応を行い、増幅曲線を得る（増幅曲線３－２’）。
　そして、得られた増幅曲線３－１’と増幅曲線３－２’を一つのグラフに表し、このグラフをホモ接合型対照とする。
　上記鋳型用混合物中の野生型コントロールと変異型コントロールは重量として等量である。 A mixture of the same wild-type control and the same mutant control as above is used as a template, and the primer pair W of the present invention is used to carry out a nucleic acid amplification reaction to obtain an amplification curve (amplification curve 3-1'). In addition, using the same wild-type control mixture as above and the same mutant control mixture as above as a template, the same nucleic acid amplification reaction is performed using the primer pair M of the present invention to obtain an amplification curve (amplification curve 3-2' ).
Then, the obtained amplification curve 3-1' and amplification curve 3-2' are plotted in one graph, and this graph is used as a homozygous control.
Wild-type and mutant controls are equal in weight in the templating mixture.
　上記の方法で得られた野生型対照の場合、増幅曲線１－１’は、増幅曲線１－２’より早く立ち上がった形となる。すなわち、増幅曲線１－１’は、増幅曲線１－２’より早期に検出される。
　ヘテロ接合型対照の場合、増幅曲線２－１’は、増幅曲線２－２’より遅く立ち上がった形となる。すなわち、増幅曲線２－１’は、増幅曲線２－２’より遅く検出される。
　ホモ接合型対照の場合、増幅曲線３－１’と増幅曲線３－２’の立ち上がりは同程度（同程度で検出される）であり、すなわち増幅曲線３－１’と増幅曲線３－２’は同程度の早ささで検出され、両増幅曲線は近接した形となる。 In the case of the wild-type control obtained by the above method, the amplification curve 1-1' rises earlier than the amplification curve 1-2'. That is, the amplification curve 1-1' is detected earlier than the amplification curve 1-2'.
In the case of the heterozygous control, the amplification curve 2-1' has a slower rise than the amplification curve 2-2'. That is, the amplification curve 2-1' is detected later than the amplification curve 2-2'.
In the case of the homozygous control, the rises of the amplification curves 3-1' and 3-2' are comparable (detected to the same extent), i.e., the amplification curves 3-1' and 3-2' are detected with similar rapidity, and the two amplification curves are close to each other.
　１－ａ－２）被検試料由来の核酸を鋳型として用いた増幅曲線の作成
　一方、被検試料由来の核酸を鋳型として用い、上記「1-a-1)対象の増幅曲線の作成」で使用したものと同じ本発明のプライマー対Ｗを用いて核酸増幅反応を行い、増幅曲線を得る（増幅曲線１）。また、同じ被検試料由来の核酸を鋳型として用い、上記「1-a-1)対象の増幅曲線の作成」で使用したものと同じ本発明のプライマー対Ｍを用いて核酸増幅反応を行い、増幅曲線を得る（増幅曲線２）。得られた増幅曲線１と増幅曲線２を一つのグラフに表す。 1-a-2) Creation of an amplification curve using a nucleic acid derived from a test sample as a template On the other hand, using a nucleic acid derived from a test sample as a template, Using the same primer pair W of the present invention as used, a nucleic acid amplification reaction is performed to obtain an amplification curve (amplification curve 1). Further, using the nucleic acid derived from the same test sample as a template, a nucleic acid amplification reaction is performed using the same primer pair M of the present invention as used in the above "1-a-1) Creation of amplification curve of object", An amplification curve is obtained (amplification curve 2). The obtained amplification curve 1 and amplification curve 2 are shown in one graph.
　１－ａ－３）塩基配列変異の判定
　上記1-a-2)で得られた被検試料由来の核酸を鋳型として用いて得られた増幅曲線１と増幅曲線２の関係を、上記1-a-1)で得られた野生型対照、ヘテロ接合型対照、ホモ接合型対照と比較する。得られた増幅曲線１と増幅曲線２の関係が野生型対照に近い場合は、被検試料の検出対象の遺伝子は野生型であると判定される。得られた増幅曲線１と増幅曲線２の関係がヘテロ接合型対照に近い場合は、被検試料の検出対象の遺伝子はヘテロ型変異であると判定される。得られた増幅曲線１と増幅曲線２の関係がホモ接合型対照に近い場合は、被検試料の検出対象の遺伝子はホモ接合型変異であると判定される。 1-a-3) Determination of nucleotide sequence mutation The relationship between the amplification curve 1 and the amplification curve 2 obtained using the nucleic acid derived from the test sample obtained in 1-a-2) above as a template is Compare with the wild-type control, heterozygous control, and homozygous control obtained in a-1). If the relationship between the obtained amplification curves 1 and 2 is close to that of the wild-type control, the gene to be detected in the test sample is determined to be wild-type. If the relationship between the obtained amplification curves 1 and 2 is close to that of the heterozygous control, it is determined that the gene to be detected in the test sample is heterozygous. If the relationship between the obtained amplification curves 1 and 2 is close to that of the homozygous control, it is determined that the gene to be detected in the test sample is homozygous mutation.
１－ｂ）その他の判定方法
　被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かを判定する別の方法としては、増幅曲線のCt値（サイクル数）やTm値を測定し、その値をもとに判定を行う方法が挙げられる。 1-b) Other determination methods Another method for determining whether the base sequence of the nucleic acid in the test sample is the wild type, homozygous mutation, or heterozygous mutation is the Ct value (cycle number) of the amplification curve, A method of measuring the Tm value and making a determination based on the value is exemplified.
　例えば、上記(i)の工程で得られた反応産物が、上記(ii)の工程で得られた反応産物よりも少ないサイクル数で検出された場合に、被検試料の検出対象の遺伝子は野生型であり、上記(i)の工程で得られた反応産物が、上記(ii)の工程で得られた反応産物よりも多いサイクル数で検出された場合に、被検試料の検出対象の遺伝子はヘテロ接合型変異であり、上記(i)の工程で得られた反応産物が、上記(ii)の工程で得られた反応産物と近接したサイクル数で検出された場合に、被検試料の検出対象の遺伝子はホモ接合型変異であると判定される。 For example, when the reaction product obtained in step (i) above is detected in a smaller number of cycles than the reaction product obtained in step (ii) above, the gene to be detected in the test sample is wild When the reaction product obtained in the above step (i) is detected in a larger number of cycles than the reaction product obtained in the above step (ii), the gene to be detected in the test sample is a heterozygous mutation, and when the reaction product obtained in step (i) above is detected at a cycle number close to the reaction product obtained in step (ii) above, the test sample The gene to be detected is determined to be a homozygous mutation.
　また上記(i)の工程の検出で得られたCt値又はTm値が、上記(ii)の工程の検出で得られたCt値又はTm値よりも大きい場合に、被検試料の検出対象の遺伝子は野生型であり、上記(i)の工程の検出で得られたCt値又はTm値が、上記(ii)の工程の検出で得られたCt値又はTm値と近似している場合に、被検試料の検出対象の遺伝子はホモ接合型変異であり、上記(i)の工程の検出で得られたCt値又はTm値が、上記(ii)の工程の検出で得られたCt値又はTm値よりも小さい場合に、被検試料の検出対象の遺伝子はヘテロ接合型変異であると判定される。 In addition, when the Ct value or Tm value obtained in the detection of the step (i) above is greater than the Ct value or Tm value obtained in the detection of the step (ii) above, When the gene is wild-type and the Ct value or Tm value obtained in the detection of the step (i) above is similar to the Ct value or Tm value obtained in the detection of the step (ii) , the gene to be detected in the test sample is a homozygous mutation, and the Ct value or Tm value obtained in the detection of the step (i) above is the Ct value obtained in the detection of the step (ii) Alternatively, when it is smaller than the Tm value, the gene to be detected in the test sample is determined to be a heterozygous mutation.
　本発明に係る被検試料の核酸の塩基配列が野生型、ホモ接合型変異、又はヘテロ接合型変異かの遺伝子型を判定する方法の一例として、インターカレーターを用いたリアルタイムPCR検出系によりα１-アンチトリプシン遺伝子変異であるPiSの遺伝子型を判定する方法を例にとって説明する。 As an example of the method of determining the genotype whether the base sequence of the nucleic acid of the test sample according to the present invention is a wild type, homozygous mutation, or heterozygous mutation, α1- by a real-time PCR detection system using an intercalator A method for determining the genotype of PiS, which is an antitrypsin gene mutation, will be described as an example.
　まず、公知の方法により、被検試料（例えばヒト血液、唾液、拭い液等）から精製DNA試料を得る。 First, a purified DNA sample is obtained from a test sample (eg, human blood, saliva, swab, etc.) by a known method.
　次に、下記のプライマー対を用い、得られた精製DNA試料を鋳型として用いて核酸増幅反応を行い（1st PCR）、精製DNAのPiS変異を含む領域を増幅させて、増幅産物を得る。
　　Ｆプライマー：5’-CTGCTGATGAAATACCTGGGCAATG-3’ （配列番号２５）
　　Ｒプライマー：5’-GGTTGGGGAATCACCTTCTGTCTTC-3’ （配列番号２６） Next, a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified DNA sample as a template (1st PCR) to amplify the region containing the PiS mutation in the purified DNA to obtain an amplified product.
F primer: 5'-CTGCTGATGAAATACCTGGGCAATG-3' (SEQ ID NO: 25)
R primer: 5'-GGTTGGGGAATCACCTTCTGTCTTC-3' (SEQ ID NO: 26)
　次に、得られた増幅産物を鋳型として用い、下記の野生型検出用プライマー対、及びPiS変異型検出用プライマー対をそれぞれ用いて、例えば下記の通りリアルタイムPCRを行う。 Next, using the obtained amplification product as a template, real-time PCR is performed, for example, as follows, using the following wild-type detection primer pair and PiS mutation type detection primer pair.
・PiS野生型検出用プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*A-3’（配列番号３５）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3’（配列番号３６）
・PiS変異型検出用プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*T-3’（配列番号３７）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3’（配列番号３８） ・ PiS wild type detection primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*A-3' (SEQ ID NO: 35)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3' (SEQ ID NO: 36)
・PiS mutation type detection primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*T-3' (SEQ ID NO: 37)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3' (SEQ ID NO: 38)
　即ち、本発明のPiS変異型検出用プライマー対又は野生型検出用プライマー対のＦプライマー及びＲプライマー各0.1～5 μM、インターカレーター（例えばEvaGreenTM（Biotium社製）、SYBRTM Green I (Molecular Probe社製）等)、夫々0.2 mM のdNTP、0.1～2単位(U)/mLのDNAポリメラーゼを含有する緩衝液又は水溶液に被検試料から精製した精製DNA試料又は１st PCRの増幅産物（10 pg～10 ng）程度を加えて調整し、PCR用反応液とする。上記PCR用反応液20 μLを、96穴反応プレートのウェルに入れ、リアルタイムPCR検出装置等を用いてリアルタイムPCRを行う。反応は30～50回サイクル繰り返し、１サイクル毎に、増幅産物に対してインターカレーションするインターカレーター由来の蛍光強度を測定する。 That is, 0.1 to 5 μM each of the F primer and the R primer of the PiS mutation type detection primer pair or the wild type detection primer pair of the present invention, an intercalator (for example, EvaGreen ™ (manufactured by Biotium), SYBR ™ Green I (Molecular Probe etc.), 0.2 mM dNTPs, 0.1 to 2 units (U)/mL of DNA polymerase, respectively, in a buffer or aqueous solution containing a purified DNA sample or 1st PCR amplification product (10 pg) purified from the test sample ~10 ng) to make the reaction mixture for PCR. 20 μL of the above reaction solution for PCR is placed in wells of a 96-well reaction plate, and real-time PCR is performed using a real-time PCR detector or the like. The reaction is repeated 30 to 50 times, and the fluorescence intensity derived from the intercalator that intercalates with the amplified product is measured for each cycle.
　次いで、得られた蛍光強度の増幅曲線を得る。 Next, obtain an amplification curve of the resulting fluorescence intensity.
　野生型検出用プライマー対を用いて得られた増幅曲線とPiS変異型検出用プライマー対を用いて得られた増幅曲線を一つの図に合わせる。 Combine the amplification curve obtained using the wild-type detection primer pair and the amplification curve obtained using the PiS mutation type detection primer pair into one figure.
　また別に、PiS変異の変異塩基配列を含む、α1-アンチトリプシン遺伝子の部分塩基配列を有するオリゴヌクレオチド（PiS変異型コントロール：例えば配列番号５４で表される塩基配列を有するオリゴヌクレオチド）、及びα1-アンチトリプシンの遺伝子のPiS変異に対応する塩基配列を含む、α1-アンチトリプシン遺伝子の野生型の塩基配列を有するオリゴヌクレオチド（PiS野生型コントロール、例えば配列番号５３で表される塩基配列を有するオリゴヌクレオチド）を得る。 Separately, an oligonucleotide having a partial nucleotide sequence of the α1-antitrypsin gene containing the mutated nucleotide sequence of the PiS mutation (PiS mutant control: for example, an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 54), and α1- Oligonucleotide having a wild-type nucleotide sequence of α1-antitrypsin gene (PiS wild-type control, for example, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 53, including a nucleotide sequence corresponding to PiS mutation in the antitrypsin gene ).
　得られたPiS変異型コントロール及びPiS野生型コントロールをそれぞれ用い、上記のPiS野生型検出用プライマー対（配列番号３５－配列番号３６）及びPiS変異型検出用プライマー対（配列番号３７－配列番号３８）を用いて、下記の方法により、野生型対照、ヘテロ接合型対照、及びホモ接合型対照の増幅曲線のグラフを得る。 Using the obtained PiS mutant control and PiS wild type control, respectively, the above PiS wild type detection primer pair (SEQ ID NO: 35-SEQ ID NO: 36) and PiS mutation detection primer pair (SEQ ID NO: 37 - SEQ ID NO: 38 ) is used to generate graphs of amplification curves for wild-type, heterozygous, and homozygous controls by the method described below.
　すなわち、野生型コントロールを鋳型として用い、PiS野生型検出用プライマー対を用いて核酸増幅反応を行って増幅曲線を得る。同じPiS野生型コントロールを鋳型として用い、PiS変異型検出用プライマー対を用いて核酸増幅反応を行って増幅曲線を得る。得られた二つの増幅曲線を一つの図に合わせ、この図を野生型対照の図とする。 That is, a wild-type control is used as a template, and a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve. Using the same PiS wild-type control as a template, a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve. The two amplification curves obtained are merged into one figure, which is the wild-type control figure.
　PiS変異型コントロールを鋳型として用い、PiS野生型検出用プライマー対を用いて核酸増幅反応を行って増幅曲線を得る。同じPiS変異型コントロールを鋳型として用い、PiS変異型検出用プライマー対を用いて核酸増幅反応を行って増幅曲線を得る。得られた二つの増幅曲線を一つの図に合わせ、この図をホモ接合型対照の図とする。 Using the PiS mutant control as a template, a nucleic acid amplification reaction is performed using a PiS wild-type detection primer pair to obtain an amplification curve. Using the same PiS mutant control as a template, a nucleic acid amplification reaction is performed using a PiS mutant detection primer pair to obtain an amplification curve. The two amplification curves obtained are combined into one figure, which is the figure of the homozygous control.
　PiS野生型コントロールとPiS変異型コントロールの混合物を鋳型として用い、PiS野生型検出用プライマー対を用いて核酸増幅反応を行って増幅曲線を得る。同じPiS野生型コントロールとPiS変異型コントロールの混合物を鋳型として用い、PiS変異型検出用プライマー対を用いて核酸増幅反応を行って得られた増幅曲線とを一つの図に合わせ、この図をヘテロ接合型対照の図とする。 A mixture of PiS wild-type control and PiS mutant control is used as a template, and a primer pair for PiS wild-type detection is used to perform a nucleic acid amplification reaction to obtain an amplification curve. Using the same mixture of PiS wild-type control and PiS mutant control as a template, the amplification curve obtained by carrying out nucleic acid amplification reaction using a pair of primers for detecting PiS mutation is combined into one figure, and this figure is used as a hetero This is a diagram of the mating type control.
　そして、被検試料由来の核酸を用いて得られた増幅曲線を、ヘテロ接合型対照、野生型対照、及びホモ接合型対照のグラフと比較する。被検試料由来の核酸を用い、PiS野生型検出用プライマー対を用いて得られた増幅曲線と、被検試料由来の核酸を用い、PiS変異型検出用プライマー対を用いて得られた増幅曲線の関係がヘテロ接合型対照と最も近い場合には、被検試料の検出対象のPiS遺伝子はヘテロ型変異であると判定する。上記増幅曲線の関係がホモ接合型対照と最も近い場合には、被検試料の検出対象のPiS遺伝子はホモ型変異であると判定する。上記増幅曲線の関係が野生型対照と最も近い場合には、被検試料の検出対象のPiS遺伝子は野生型であると判定する。 Then, the amplification curves obtained using the test sample-derived nucleic acids are compared with the heterozygous control, wild-type control, and homozygous control graphs. An amplification curve obtained using a nucleic acid derived from a test sample and using a PiS wild-type detection primer pair, and an amplification curve obtained using a nucleic acid derived from a test sample and using a PiS mutation type detection primer pair is closest to that of the heterozygous control, the PiS gene to be detected in the test sample is determined to be heterozygous. If the relationship between the amplification curves is the closest to that of the homozygous control, the PiS gene to be detected in the test sample is determined to be homozygous mutation. If the relationship between the amplification curves is closest to that of the wild-type control, the PiS gene to be detected in the test sample is determined to be wild-type.
２）被検試料の核酸の塩基配列が野生型か変異型かを判定する方法について 2) Regarding the method for determining whether the base sequence of the nucleic acid in the test sample is wild-type or mutant
　本発明のプライマー対Ｍ又は／及び本発明のプライマー対Ｗを用い、被検試料由来の核酸を鋳型として用い、核酸増幅反応を行う。
　被検試料の検出対象の遺伝子の塩基配列が変異型である場合には、本発明のプライマー対Ｍを用いた核酸増幅反応では反応産物が検出されるが、本発明のプライマー対Ｗを用いた核酸増幅反応では反応産物が検出されないか、ほとんど検出されない。
　被検試料の検出対象の遺伝子の塩基配列が野生型である場合には、本発明の本発明のプライマー対Ｍを用いた核酸増幅反応では反応産物が検出されない、又はほとんど検出されない。一方、本発明のプライマー対Ｗを用いた核酸増幅反応では反応産物が検出される。 A nucleic acid amplification reaction is performed using the primer pair M of the present invention and/or the primer pair W of the present invention, and using nucleic acid derived from a test sample as a template.
When the base sequence of the gene to be detected in the test sample is a mutant, the reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention, but the reaction product is detected using the primer pair W of the present invention. No or very little reaction product is detected in nucleic acid amplification reactions.
When the nucleotide sequence of the gene to be detected in the test sample is of the wild type, no or almost no reaction product is detected in the nucleic acid amplification reaction using the primer pair M of the present invention. On the other hand, a reaction product is detected in the nucleic acid amplification reaction using the primer pair W of the present invention.
　例えば本発明のプライマー対Ｍと、インターカレーターを用い、被検試料から精製した精製DNA試料を鋳型として用いて、リアルタイムPCRを行う。そして増幅産物に対してインターカレーションするインターカレーター由来の蛍光強度を測定する。
　また、本発明のプライマー対Ｗを用いる以外は本発明のプライマー対Ｍを用いた場合と同じ方法でリアルタイムPCRを行い、インターカレーター由来の蛍光強度を測定する。 For example, real-time PCR is performed using the primer pair M of the present invention, an intercalator, and a purified DNA sample purified from a test sample as a template. Then, the fluorescence intensity derived from the intercalator that intercalates with the amplified product is measured.
Real-time PCR is performed in the same manner as in the case of using the primer pair M of the present invention except that the primer pair W of the present invention is used, and the fluorescence intensity derived from the intercalator is measured.
　そして、本発明のプライマー対Ｍを用いた核酸増幅反応では反応産物が得られたが、本発明のプライマー対Ｗを用いた核酸増幅反応では反応産物が検出されないか、ほとんど検出されなかった場合には、その被検試料の検出対象の遺伝子の塩基配列は、目的の遺伝子の塩基配列変異を有する、と判定される。
　一方、例えば本発明のプライマー対Ｍを用いた核酸増幅反応では反応産物が検出されないか、ほとんど検出されなかったが、本発明のプライマー対Ｗを用いた核酸増幅反応では反応産物が得られた場合には、その被検試料の検出対象の遺伝子の塩基配列は、目的の遺伝子の塩基配列変異を有さない、と判定される。 Then, in the nucleic acid amplification reaction using the primer pair M of the present invention, a reaction product was obtained, but in the nucleic acid amplification reaction using the primer pair W of the present invention, no or almost no reaction product was detected. , it is determined that the base sequence of the gene to be detected in the test sample has a base sequence mutation of the target gene.
On the other hand, for example, in the nucleic acid amplification reaction using the primer pair M of the present invention, no or almost no reaction product was detected, but in the nucleic acid amplification reaction using the primer pair W of the present invention, a reaction product was obtained. First, it is determined that the base sequence of the gene to be detected in the test sample does not have the base sequence mutation of the target gene.
　本発明に係る被検試料の核酸の塩基配列が野生型か変異型かを判定する方法の一例として、SARS-CoV-2ウイルスのN501Y変異を判定する方法を例にとって説明する。 As an example of the method for determining whether the nucleic acid sequence of a test sample according to the present invention is wild-type or mutant, the method for determining the N501Y mutation of the SARS-CoV-2 virus will be described.
　まず、公知の方法により、SARS-CoV-2ウイルスから精製total RNAを得、得られた一本鎖RNAを鋳型として用い、常法による逆転写反応を行ってcDNAを得る。 First, purified total RNA is obtained from the SARS-CoV-2 virus by a known method, and using the obtained single-stranded RNA as a template, reverse transcription is performed by a conventional method to obtain cDNA.
　次に、下記のプライマー対を用い、得られた精製cDNAを鋳型として用いて核酸増幅反応を行い（1st PCR）、cDNAのN501Y変異を含む領域を増幅させて、増幅産物を得る。
　　Fプライマー：5’-CTATCAGGCCGGTAGCACACCTTG-3’ （配列番号７５）
　　Rプライマー：5’-CCACAAACAGTTGCTGGTGCATGTAG-3’ （配列番号７６） Next, a nucleic acid amplification reaction is performed using the following primer pair and the resulting purified cDNA as a template (1st PCR) to amplify the region containing the N501Y mutation in the cDNA to obtain an amplified product.
F primer: 5'-CTATCAGGCCGGTAGCACACCTTG-3' (SEQ ID NO: 75)
R primer: 5'-CCACAAACAGTTGCTGGTGCATGTAG-3' (SEQ ID NO: 76)
　次に、得られた増幅産物を鋳型として用い、下記のN501Y野生型検出用プライマー対、及びN501Y変異型検出用プライマー対をそれぞれ用いて、例えばインターカレーター法によるリアルタイムPCRを行う。 Next, using the obtained amplification product as a template, real-time PCR is performed, for example, by the intercalator method, using the following N501Y wild-type detection primer pair and N501Y mutant-type detection primer pair.
・N501Y野生型検出用プライマー対
　　Fプライマー：5’-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3’ （配列番号７９）
　　Rプライマー：5’-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3’ （配列番号８０）
・N501Y変異型検出用プライマー対
　　Fプライマー：5’-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3’ （配列番号８１）
　　Rプライマー：5’-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3’ （配列番号８２） ・N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79)
R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80)
・N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81)
R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
　そして、N501Y変異型検出用プライマー対（本発明のプライマー対Ｍ）を用いた核酸増幅反応では反応産物が得られたが、N501Y野生型検出用プライマー対（本発明のプライマー対Ｗ）を用いた核酸増幅反応では反応産物が検出されないか、ほとんど検出されなかった場合には、その被検試料の検出対象のN501Y遺伝子の塩基配列は、N501Y変異を有する、と判定される。
　一方、N501Y変異型検出用プライマー対（本発明のプライマー対Ｍ）を用いた核酸増幅反応では反応産物が検出されないか、ほとんど検出されなかったが、N501Y野生型検出用プライマー対（本発明のプライマー対Ｗ）を用いた核酸増幅反応では反応産物が得られた場合には、その被検試料の検出対象のN501Y遺伝子の塩基配列は変異を有さない野生型である、と判定される。 Then, a reaction product was obtained in the nucleic acid amplification reaction using the N501Y mutant detection primer pair (primer pair M of the present invention), but the N501Y wild type detection primer pair (primer pair W of the present invention) was used. When no reaction product or almost no reaction product is detected in the nucleic acid amplification reaction, it is determined that the base sequence of the N501Y gene to be detected in the test sample has the N501Y mutation.
On the other hand, in the nucleic acid amplification reaction using the primer pair for detecting N501Y mutant type (primer pair M of the present invention), reaction products were not detected or hardly detected, but the primer pair for detecting N501Y wild type (primer pair of the present invention If a reaction product is obtained in the nucleic acid amplification reaction using pair W), it is determined that the nucleotide sequence of the N501Y gene to be detected in the test sample is of the wild type without mutation.
　尚、本発明の変異を判定する方法においてプライマー対Ｍ及びプライマー対Ｗを用いる場合、使用するプライマー対ＭのＦプライマーとプライマー対ＷのＦプライマーは、「第２鎖の変異塩基配列の相補配列」に相当する配列以外の塩基配列は、同じであることが好ましい。
　同様に、使用するプライマー対ＭのＲプライマーとプライマー対ＷのＲプライマーは、「第１鎖の変異塩基配列の相補配列」に相当する配列以外の塩基配列は、同じであることが好ましい。 In addition, when primer pair M and primer pair W are used in the method for determining mutation of the present invention, the F primer of primer pair M and the F primer of primer pair W to be used are "complementary sequence of the mutated nucleotide sequence of the second strand ” are preferably the same.
Similarly, the R primer of the primer pair M and the R primer of the primer pair W to be used preferably have the same base sequence except for the sequence corresponding to the "complementary sequence of the mutated base sequence of the first strand."
　また、プライマー対Ｍ及びプライマー対Ｗを用いる場合、使用するプライマー対ＭのＦプライマーとプライマー対ＷのＦプライマーのＳ化されたヌクレオチドの数は、同じであることが好ましい。同様に、使用するプライマー対ＭのＲプライマーとプライマー対ＷのＲプライマーのＳ化されたヌクレオチドの数は、同じであることが好ましい。 In addition, when primer pair M and primer pair W are used, it is preferable that the number of S-nucleotides in the F primer of primer pair M and the F primer of primer pair W to be used is the same. Similarly, the number of s-nucleotides in the R primer of primer pair M and the R primer of primer pair W to be used is preferably the same.
＜３．本発明の塩基配列の変異判定用キット＞
　本発明のキットは、被検試料の核酸の塩基配列が野生型、ホモ接合型変異、若しくはヘテロ接合型変異の判定用、或いは野生型又は変異型の判定用キットである。 <3. Nucleotide sequence mutation determination kit of the present invention>
The kit of the present invention is a kit for determining whether the base sequence of a nucleic acid in a test sample is wild-type, homozygous mutation, or heterozygous mutation, or for determining whether it is wild-type or mutant.
　本発明のキットにおいて塩基配列の変異を検出し判定する対象の遺伝子（変異の形態）の例としては上記したものが挙げられ、より具体的には、例えばヒトの場合のBRAF，α1－アンチトリプシン（Pis、PiZ）等、SARS-CoV-2の場合のN501Y，E484K等が挙げられる。 Examples of target genes (mutation forms) for detection and determination of nucleotide sequence mutations in the kit of the present invention include those described above. (Pis, PiZ), etc., and N501Y, E484K, etc. in the case of SARS-CoV-2.
　本発明に係る塩基配列の変異判定用キットとしては、本発明のプライマー対を含むものが挙げられる。好ましくは、本発明のプライマー対Ｍ及び／又は本発明のプライマー対Ｗを含むものが挙げられる。本発明のプライマーＭ及び本発明のプライマーＷを含むものが好ましい。
　本発明のキットに係る本発明のプライマー対、本発明のプライマー対Ｍ及び本発明のプライマー対Ｗについては、「＜１．本発明のプライマー対＞」の項で説明した通りであり、好ましい例、具体例等も同じである。 Examples of the nucleotide sequence mutation determination kit according to the present invention include those containing the primer pair of the present invention. Preferred are those containing the primer pair M of the present invention and/or the primer pair W of the present invention. Those containing the primer M of the present invention and the primer W of the present invention are preferred.
The primer pair of the present invention, the primer pair M of the present invention, and the primer pair W of the present invention according to the kit of the present invention are as described in the section "<1. Primer pair of the present invention>", and preferred examples , specific examples, etc. are also the same.
　本発明のキットに係る本発明のプライマー対は、適当な緩衝液中に懸濁させた懸濁液等の溶液状態の試液での形態であってもよく、凍結品や凍結乾燥品であってもよい。
　本発明のプライマー対の上記試液中の濃度、試液の溶媒等は、通常この分野で使用される範囲で適宜設定されればよい。 The primer pair of the present invention according to the kit of the present invention may be in the form of a solution-state test solution such as a suspension suspended in an appropriate buffer, or may be a frozen product or a lyophilized product. good too.
The concentration of the primer pair of the present invention in the test solution, the solvent of the test solution, and the like may be appropriately set within the range usually used in this field.
　本発明のキットには、核酸増幅反応を行うために必要な試薬を必要量備えていてもよい。
　例えば、本発明のプライマー対の他に、更にヌクレオシド三リン酸、核酸合成酵素、PCR緩衝液等を備えていてもよい。 The kit of the present invention may contain necessary amounts of reagents necessary for carrying out a nucleic acid amplification reaction.
For example, in addition to the primer pair of the present invention, nucleoside triphosphate, nucleic acid synthetase, PCR buffer and the like may be further provided.
　上記の核酸合成酵素としては、例えばDNAポリメラーゼ、RNAポリメラーゼ、逆転写酵素等が挙げられる。 Examples of the above nucleic acid synthetase include DNA polymerase, RNA polymerase, reverse transcriptase, and the like.
　上記のPCR緩衝液として用いられる緩衝液の具体例としては、例えばトリス緩衝液、リン酸緩衝液、ベロナール緩衝液、ホウ酸緩衝液、グッド緩衝液等、通常のPCRやハイブリダイゼーション反応を実施する場合に用いられている緩衝液は全て挙げられ、そのpHも特に限定されない。 Specific examples of buffers used as the PCR buffer include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, etc. All the buffers used in the case are listed, and the pH thereof is not particularly limited.
　また、本発明のキットには、必要に応じて酵素に応じた基質（dNTP、rNTPなど）、また例えば例えばEvaGreenTM（コスモ・バイオ(株)製），SYBRTM Green I (Molecular Probe社製)，エチジウムブロマイド，フルオレン等の二本鎖インターカレーター、FAMやTAMRA等の標識検出物質などを含んでいてもよい。また、例えば安定化剤、防腐剤等であって、共存する試薬等の安定性を阻害せず、PCRやハイブリダイゼーション反応を阻害しないものが含まれていてもよい。また、これらの濃度も、通常この分野で通常用いられる濃度範囲から適宜選択すればよい。 In addition, the kit of the present invention may optionally contain substrates (dNTPs, rNTPs, etc.) suitable for the enzyme, such as EvaGreen ™ (manufactured by Cosmo Bio Co., Ltd.), SYBR ™ Green I (manufactured by Molecular Probe). , ethidium bromide, fluorene, and other double-stranded intercalators, and labeled detection substances such as FAM and TAMRA. In addition, for example, stabilizers, preservatives, etc., which do not inhibit the stability of coexisting reagents and the like, and which do not inhibit PCR and hybridization reactions, may be included. Moreover, these concentrations may also be appropriately selected from the concentration ranges normally used in this field.
　また、本発明のキットには、検出対象の遺伝子の変異塩基配列を含む、上記遺伝子の塩基配列（部分配列でよい）を有するオリゴヌクレオチド（野生型コントロール）及び／又は上記遺伝子の野生型の塩基配列配列を含み、且つ上記変異に対応する塩基配列を含むオリゴヌクレオチド（変異型コントロール）を含んでいてもよい。
　また本発明のキットは遺伝子型（野生型、ヘテロ接合型、又はホモ接合型）を判定するための増幅曲線の見本を含んでいてもよい。
　また、本発明のキットは、上記した１st PCRに用いられるプライマー対、及び１st PCRに必要な試薬を必要量備えていてもよい。 In addition, the kit of the present invention contains an oligonucleotide (wild-type control) having the base sequence of the gene (which may be a partial sequence), including the mutated base sequence of the gene to be detected, and/or the wild-type base of the gene. An oligonucleotide (mutant type control) containing the sequence sequence and the base sequence corresponding to the mutation may be included.
Kits of the invention may also include sample amplification curves for determining genotype (wild-type, heterozygous, or homozygous).
In addition, the kit of the present invention may comprise the necessary amount of the primer pair used in the 1st PCR and the reagents necessary for the 1st PCR.
　更にまた本発明のキットには、本発明のプライマーを用いた核酸増幅反応の手順、野生型コントロール及び変異型コントロールを用いて遺伝子型（野生型、ヘテロ接合型、又はホモ接合型）を判定するための対照となる増幅曲線を作成する手順、作成した対照となる増幅曲線又は本キットに添付の増幅曲線の見本をもとに塩基配列変異を判定する手順、等を説明した説明書等を含ませておいても良い。上記「説明書」とは、上記方法における特徴・原理・操作手順、判定手順等が文章又は図表等により実質的に記載されている上記キットの取扱説明書、添付文書、あるいはパンフレット（リーフレット）等を意味する。 Furthermore, the kit of the present invention includes a nucleic acid amplification reaction procedure using the primers of the present invention, a wild-type control and a mutant control to determine the genotype (wild-type, heterozygous, or homozygous). Instructions, etc., explaining the procedure for preparing an amplification curve to be used as a control for testing, the procedure for determining base sequence mutations based on the prepared control amplification curve or the sample amplification curve attached to this kit, etc. You can let it go. The above-mentioned "instruction" means the instruction manual, package insert, pamphlet (leaflet), etc. of the above-mentioned kit in which the features, principles, operation procedures, judgment procedures, etc. of the above-mentioned method are substantially described in sentences, diagrams, etc. means
　更にまた本発明のキットは、被検試料を採取するための手段（例えば綿棒等）を備えていてもよい。 Furthermore, the kit of the present invention may be equipped with means (for example, cotton swabs, etc.) for collecting test samples.
　以下に実施例及び比較例を挙げて、本発明を更に具体的に説明するが、本発明はこれらにより何等限定されるものではない。 The present invention will be described in more detail with reference to examples and comparative examples below, but the present invention is not limited by these.
　尚、実施例及び比較例に於いて、塩基配列の塩基として示した「Ａ」又は「a」はアデニンを、「Ｇ」又は「g」はグアニンを、「Ｃ」又は「c」はシトシンを、「Ｔ」又は「t」はチミンを夫々示す。 In Examples and Comparative Examples, "A" or "a" shown as a base in the base sequence is adenine, "G" or "g" is guanine, and "C" or "c" is cytosine. , "T" or "t" indicate thymine, respectively.
実施例１．ヒトBRAF遺伝子の１塩基変異検出
　以下の方法で、本発明のヒトBRAF遺伝子の１塩基置換（c1799T＞A(V600E)）の検出を行った。
（１）ゲノムDNAの抽出
　TIG-3（ヒト胎児正常線維芽細胞、JCRB細胞バンク）、COLO201（ヒト結腸腺癌細胞、ATCC No. CCL-224）及びA375（メラノーマ細胞、ATCC No. CRL-1619）（1×106）の培養液を遠心分離後、上清を除去後、細胞を回収した。QuickGene SP kit DNA tissue（倉敷紡績(株)）を用い、同キットのプロトコールに従って、回収した細胞中のゲノムDNAを抽出した。 Example 1. Detection of Single Nucleotide Mutation in Human BRAF Gene A single nucleotide substitution (c1799T>A(V600E)) in the human BRAF gene of the present invention was detected by the following method.
(1) Genomic DNA extraction TIG-3 (human fetal normal fibroblasts, JCRB cell bank), COLO201 (human colon adenocarcinoma cells, ATCC No. CCL-224) and A375 (melanoma cells, ATCC No. CRL-1619) ) (1×10 6 ) of the culture solution was centrifuged, the supernatant was removed, and the cells were collected. Using QuickGene SP kit DNA tissue (Kurashiki Spinning Co., Ltd.), genomic DNA in the collected cells was extracted according to the protocol of the kit.
（２）リアルタイムPCR
１）プライマー対
　下記のプライマー対を設計し、合成した。
　尚、各プライマーの塩基配列において、*印はホスホジエステル結合がＳ化された位置を示す。 (2) Real-time PCR
1) Primer pair The following primer pair was designed and synthesized.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
i)Ｓ化なしプライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGT-3’（配列番号１）
　Ｒプライマー: 5’-CAAACTGATGGGACCCACTCCATCGAGATTTCA-3’ （配列番号２）
・変異型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA-3’ （配列番号３）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGATTTCT-3’ （配列番号４） i) Primer pair without S-merization/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGT-3' (SEQ ID NO: 1)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCA-3' (SEQ ID NO: 2)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA-3' (SEQ ID NO: 3)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTCT-3' (SEQ ID NO: 4)
ii)１塩基Ｓ化プライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG*T-3’ （配列番号５）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGATTTC*A-3’ （配列番号６）
・変異型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG*A-3’ （配列番号７）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGATTTC*T-3’ （配列番号８） ii) 1 base S-primer pair/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG*T-3' (SEQ ID NO: 5)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTC*A-3' (SEQ ID NO: 6)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAG*A-3' (SEQ ID NO: 7)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTTC*T-3' (SEQ ID NO: 8)
iii)２塩基Ｓ化プライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACA*G*T-3’（配列番号９）
　Ｒプライマー： 5’-CAAACTGATGGGACCCACTCCATCGAGATTT*C*A-3’ （配列番号１０）
・変異型検出用プライマー対
　Ｆプライマー：5‘-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACA*G*A-3’ （配列番号１１）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGATTT*C*T-3’ （配列番号１２） iii) 2 base S-primer pair/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACA*G*T-3' (SEQ ID NO: 9)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTT*C*A-3' (SEQ ID NO: 10)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACA*G*A-3' (SEQ ID NO: 11)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATTT*C*T-3' (SEQ ID NO: 12)
iv)３塩基Ｓ化プライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTAC*A*G*T-3’ （配列番号１３）
　Ｒプライマー： 5’-CAAACTGATGGGACCCACTCCATCGAGATT*T*C*A-3’ （配列番号１４）
・変異型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTAC*A*G*A-3’ （配列番号１５）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGATT*T*C*T-3’ （配列番号１６） iv) 3-base S-primer pair/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTAC*A*G*T-3' (SEQ ID NO: 13)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATT*T*C*A-3' (SEQ ID NO: 14)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTAC*A*G*A-3' (SEQ ID NO: 15)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGATT*T*C*T-3' (SEQ ID NO: 16)
v)４塩基Ｓ化プライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTA*C*A*G*T-3’（配列番号１７）
　Ｒプライマー： 5’-CAAACTGATGGGACCCACTCCATCGAGAT*T*T*C*A-3’ （配列番号１８）
・変異型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTA*C*A*G*A-3’ （配列番号１９）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGAT*T*T*C*T-3’ （配列番号２０） v) 4-base S-primer pair/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTA*C*A*G*T-3' (SEQ ID NO: 17)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGAT*T*T*C*A-3' (SEQ ID NO: 18)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCTA*C*A*G*A-3' (SEQ ID NO: 19)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGAT*T*T*C*T-3' (SEQ ID NO: 20)
vi)５塩基Ｓ化プライマー対
・野生型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCT*A*C*A*G*T-3’ （配列番号２１）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGA*T*T*T*C*A-3’ （配列番号２２）
・変異型検出用プライマー対
　Ｆプライマー：5’-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCT*A*C*A*G*A-3’ （配列番号２３）
　Ｒプライマー：5’-CAAACTGATGGGACCCACTCCATCGAGA*T*T*T*C*T-3’ （配列番号２４） vi) 5-base S-primer pair/primer pair for wild-type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCT*A*C*A*G*T-3' (SEQ ID NO: 21)
R primer: 5'-CAAACTGATGGGACCCCACTCCATCGAGA*T*T*T*C*A-3' (SEQ ID NO: 22)
・Primer pair for mutation type detection F primer: 5'-CTCACAGTAAAAATAGGTGATTTTGGTCTAGCT*A*C*A*G*A-3' (SEQ ID NO: 23)
R primer: 5'-CAAACTGATGGGACCCACTCCATCGAGA*T*T*T*C*T-3' (SEQ ID NO: 24)
　ヒトBRAF遺伝子の１塩基変異（c1799T＞A(V600E)）は、ヒトBRAF遺伝子の塩基配列の1799位のTがAに変異している。 A single-nucleotide mutation in the human BRAF gene (c1799T>A (V600E)) is a mutation of T to A at position 1799 in the nucleotide sequence of the human BRAF gene.
　ヒトBRAF遺伝子の二本鎖核酸において、その変異塩基Aを有する側の鎖を第１鎖とし、その相補鎖を第２鎖とした場合、本実施例１で用いた変異型検出用プライマー対のＲプライマーは、第１鎖の変異塩基Aの相補塩基Tを３'末端に有する。Ｒプライマーのその他の塩基配列は、上記第１鎖の上記変異塩基Tに隣接する３’側の塩基配列の相補配列と同じである。
　また、上記変異型検出用プライマー対のＦプライマーは、第２鎖の変異塩基Tの相補塩基Aを３'末端に有する。Ｆプライマーのその他の塩基配列は、上記第２鎖の上記変異塩基Tに隣接する３’側の塩基配列の相補配列と同じである。 In the double-stranded nucleic acid of the human BRAF gene, when the strand on the side having the mutated base A is the first strand and the complementary strand is the second strand, the mutation detection primer pair used in Example 1 is The R primer has the complementary base T of the mutated base A of the first strand at the 3' end. The rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
In addition, the F primer of the pair of primers for detecting mutation type has a complementary base A of the mutated base T of the second strand at the 3' end. The rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
２）リアルタイムPCR
　上記(１)で得られたゲノムDNA 100pgを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)製）を用い、プライマー対として上記１)で得られた各プライマー対を用いて、以下の方法でリアルタイムPCRを行った 2) Real-time PCR
Using 100 pg of genomic DNA obtained in (1) above as a template, using KOD-FX-Neo (manufactured by Toyobo Co., Ltd.) as a thermostable DNA polymerase, and using each primer pair obtained in (1) above as a primer pair. Real-time PCR was performed using the following method.
ｉ）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 i) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000017
EvaGreenTM(Biotium社製)：定量リアルタイムPCR用蛍光試薬、コスモ・バイオ(株)製
KOD FX Neo（東洋紡(株)製）：1 U/μL
Figure JPOXMLDOC01-appb-I000017
EvaGreen TM (manufactured by Biotium): fluorescent reagent for quantitative real-time PCR, manufactured by Cosmo Bio Co., Ltd.
KOD FX Neo (manufactured by Toyobo Co., Ltd.): 1 U/μL
ii）リアルアイムPCR
　上記ｉ）で調製したPCR用反応液20μＬを、９６ 穴反応プレート（マイクロアンプ・オプチカル・９６ウェル・リアクション・プレート、アプライドバイオシステムズジャパン社製）のウェルに入れ、TaqManTM　PCR 専用サーマルサイクラー・検出器（ABI 7500、アプライドバイオシステムズジャパン社製） を用いてリアルタイムPCRを行った。即ち、94℃で2分間保温の後、98℃で5秒間、68℃で10秒間の反応を40サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 ii) real-time PCR
20 μL of the reaction solution for PCR prepared in i) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well reaction plate, manufactured by Applied Biosystems Japan Co., Ltd.) and subjected to TaqMan ™ PCR thermal cycler detection. Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（３）結果
　得られた増幅曲線を図１～図６に示す。
　図１はＳ化していないプライマーのプライマー対を用いた結果を示す。
　図２は１塩基がＳ化されたプライマーのプライマー対を用いた結果を示す。
　図３は２塩基がＳ化されたプライマーのプライマー対を用いた結果を示す。
　図４は３塩基がＳ化されたプライマーのプライマー対を用いた結果を示す。
　図５は４塩基がＳ化されたプライマーのプライマー対を用いた結果を示す。
　図６は５塩基がＳ化されたプライマーのプライマー対を用いた結果を示す。 (3) Results The obtained amplification curves are shown in FIGS. 1 to 6. FIG.
FIG. 1 shows the results using a primer pair of primers that are not S-conjugated.
FIG. 2 shows the results of using a primer pair of primers in which one nucleotide is S-converted.
FIG. 3 shows the results of using a primer pair of primers in which 2 bases are S-converted.
FIG. 4 shows the results of using a primer pair of primers in which 3 nucleotides are S-converted.
FIG. 5 shows the results of using a primer pair of primers in which 4 bases are S-converted.
FIG. 6 shows the results of using a primer pair of primers in which 5 nucleotides are S-converted.
　また図１～図６において、（１）はTIG-3のゲノムDNAを鋳型として用いた結果を、（２）はCOLO201のゲノムDNAを鋳型として用いた結果を、（３）はA375のゲノムDNAを鋳型として用いた結果をそれぞれ示す。 In FIGS. 1 to 6, (1) shows the results of using TIG-3 genomic DNA as a template, (2) shows the results of using COLO201 genomic DNA as a template, and (3) shows the results of A375 genomic DNA. are used as templates.
　更に、図１～図６において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線Ｍは変異型検出用プライマー対を用いた結果をそれぞれ示す。 Furthermore, in FIGS. 1 to 6, the amplification curve W shows the results of using the wild-type detection primer pair, and the amplification curve M shows the results of using the mutation-type detection primer pair.
　TIG-3細胞は、BRAF遺伝子の変異がない。COLO201細胞は、BRAF遺伝子のc1799T＞Aのヘテロ接合型変異を持つ。A375細胞は、BRAF遺伝子のc1799T＞Aのホモ接合型変異を持つ。 　TIG-3 cells do not have mutations in the BRAF gene. COLO201 cells have a heterozygous mutation c1799T>A in the BRAF gene. A375 cells have a homozygous c1799T>A mutation in the BRAF gene.
　Ｓ化されていないプライマーを用いた検出（図１）、及び１塩基がＳ化されたプライマーを用いた検出（図２）では、ヘテロ接合型（(2)COLO201）とホモ接合型（(3)A375）との間に増幅曲線の明確な違いはみられなかった。 In the detection using a primer that is not S-conjugated (Fig. 1) and the detection using a primer with one base S-conjugated (Fig. 2), the heterozygous type ((2) COLO201) and the homozygous type ((3) )A375) did not show a clear difference in the amplification curves.
　２塩基がＳ化されたプライマーを用いた検出（図３）、３塩基がＳ化されたプライマーを用いた検出（図４）、４塩基がＳ化されたプライマーを用いた検出（図５）、及び５塩基がＳ化されたプライマーを用いた検出（図６）では、野生型（(1)TIG-3）では野生型検出用プライマー対を用いた増幅曲線が変異型検出用プライマー対を用いた増幅曲線よりも先に立ち上がっていた（より早期に検出された）。一方、ヘテロ接合型（(2)COLO201）では野生型検出用プライマー対を用いた増幅曲線と変異型検出用プライマー対を用いた増幅曲線が近接していた。また、ホモ接合型（(3)A375）では野生型（(1)TIG-3）とは逆に、変異型検出用プライマー対を用いた増幅曲線の方が野生型検出用プライマー対を用いた増幅曲線よりも先に立ち上がっていた（より早期に検出された）。 Detection using a primer having two S-bases (Fig. 3), detection using a primer having three S-bases (Fig. 4), detection using a primer having four S-bases (Fig. 5) , and detection using primers in which 5 bases are S-converted (FIG. 6), the amplification curve using the wild type detection primer pair for the wild type ((1) TIG-3) shows the mutation type detection primer pair It rose earlier (earlier detection) than the amplification curve used. On the other hand, in the heterozygous type ((2) COLO201), the amplification curve using the wild-type detection primer pair and the amplification curve using the mutant detection primer pair were close to each other. In contrast to the wild type ((1) TIG-3), the homozygous type ((3) A375) used the wild type detection primer pair in the amplification curve using the mutant type detection primer pair. It rose ahead of the amplification curve (detected earlier).
　以上のことから、２～５塩基がＳ化されたプライマーのプライマー対を用いた検出を行うことにより、野生型と変異型の区別のみならず、ヘテロ接合型とホモ接合型を区別して判定できることが明らかとなった。 From the above, by performing detection using a primer pair of primers in which 2 to 5 bases are S-modified, it is possible to distinguish not only between wild type and mutant type, but also between heterozygous type and homozygous type. became clear.
　特に、２塩基がＳ化されたプライマーのプライマー対又は３塩基がＳ化されたプライマーのプライマー対を用いた検出を行うことにより、ヘテロ接合型とホモ接合型を明確に区別して判定できることがわかった。 In particular, it was found that the heterozygous type and the homozygous type can be clearly distinguished and determined by performing detection using a primer pair of primers in which 2 bases are S-converted or a primer pair of primers in which 3 bases are S-converted. rice field.
実施例２．ヒトα1-アンチトリプシンの変異の検出－１(PiS)
　α1-アンチトリプシン欠乏症のヒトでは、以下の遺伝子の１塩基置換が知られている。
　　　PiS :  c.863A>T p.Glu288Val 
　　　PiZ :  c.1096G>A p.Glu366Lys
　実施例２では、本発明のプライマー対を用い、PiS変異の検出を行った。 Example 2. Mutation detection of human α1-antitrypsin-1 (PiS)
Humans with α1-antitrypsin deficiency are known to have the following single base substitutions in the gene.
PiS : c.863A>T p.Glu288Val
PiZ : c.1096G>A p.Glu366Lys
In Example 2, PiS mutation was detected using the primer pair of the present invention.
（１）ゲノムDNAの抽出
　COLO201（ヒト結腸癌細胞、ATCC No. CCL-224）、A549（ヒト肺癌細胞、ATCC No. CCL-185）、HepG2（ヒト肝癌細胞、ATCC No. HB-8065）及びMCF7（ヒト乳癌細胞、ATCC No. HTB-22）の培養液を遠心分離し、上清を除去後、細胞を回収した。QuickGene SP kit DNA tissue（倉敷紡績(株)）を用い、同キットのプロトコールに従って、回収した細胞中のゲノムDNAを抽出した。 (1) Genomic DNA extraction COLO201 (human colon cancer cells, ATCC No. CCL-224), A549 (human lung cancer cells, ATCC No. CCL-185), HepG2 (human liver cancer cells, ATCC No. HB-8065) and A culture solution of MCF7 (human breast cancer cells, ATCC No. HTB-22) was centrifuged, and after removing the supernatant, the cells were collected. Using QuickGene SP kit DNA tissue (Kurashiki Spinning Co., Ltd.), genomic DNA in the collected cells was extracted according to the protocol of the kit.
（２）1st PCR
　上記（１）で抽出した各細胞のゲノムDNA（それぞれn=2） 100pgを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (2) 1st PCR
Using 100 pg of genomic DNA (n=2 each) of each cell extracted in (1) above as a template, using KOD-Plus-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, PCR amplification reaction was carried out in the following manner. did
１）ゲノムDNAのPCR増幅用プライマー対
　下記のプライマー対を設計し、合成した。
　　Ｆプライマー：5’-CTGCTGATGAAATACCTGGGCAATG-3’ （配列番号２５）
　　Ｒプライマー：5’-GGTTGGGGAATCACCTTCTGTCTTC-3’ （配列番号２６） 1) Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
F primer: 5'-CTGCTGATGAAATACCTGGGCAATG-3' (SEQ ID NO: 25)
R primer: 5'-GGTTGGGGAATCACCTTCTGTCTTC-3' (SEQ ID NO: 26)
２）PCR反応液の調整
　下記の組成のPCR反応液を調整した。
Figure JPOXMLDOC01-appb-I000018
KOD Plus Neo：1 U/μL 2) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000018
KOD Plus Neo: 1 U/μL
３）1st PCR
　上記２)で調製したPCR用反応液20μＬを、９６ 穴反応プレート（マイクロアンプ・オプチカル・９６ウェル・リアクション・プレート、アプライドバイオシステムズジャパン社製）のウェルに入れ、TaqManTM　PCR 専用サーマルサイクラー・検出器（ABI 7500、アプライドバイオシステムズジャパン社製）を用いてPCRを行い、PCR増幅産物を得た。
　PCRは、94℃で2分間保温の後、98℃で8秒間、68℃で20秒間の反応を36サイクル繰り返した。
　この1st-PCRでは、α1-アンチトリプシン遺伝子のPiS変異が起きる位置である863位の塩基を含む領域が増幅される。 3) 1st PCR
20 μL of the reaction solution for PCR prepared in 2) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well Reaction Plate, Applied Biosystems Japan), and a thermal cycler for TaqMan ™ PCR is used for detection. PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan) to obtain a PCR amplification product.
PCR was carried out by incubating at 94°C for 2 minutes, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
This 1st-PCR amplifies a region containing the 863rd base, which is the position where PiS mutation occurs in the α1-antitrypsin gene.
（３）リアルタイムPCR
１）Ｓ化プライマー対
　下記の３個のヌクレオチドがＳ化されたプライマーのプライマー対を設計し、合成した。
　尚、各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 (3) Real-time PCR
1) S-Primer Pairs The following primer pairs in which three nucleotides were S-substituted were designed and synthesized.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
・PiS野生型検出用プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*A-3’（配列番号３５）
　Ｒプライマー: 5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3’（配列番号３６）
・PiS変異型検出用プライマー対
　Ｆプライマー：5’-GATGAGGGGAAACTACAGCACCT*G*G*T-3’（配列番号３７）
　Ｒプライマー：5’-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3’（配列番号３８） ・ PiS wild type detection primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*A-3' (SEQ ID NO: 35)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*T-3' (SEQ ID NO: 36)
・PiS mutation type detection primer pair F primer: 5'-GATGAGGGGAAACTACAGCACCT*G*G*T-3' (SEQ ID NO: 37)
R primer: 5'-GTGATGATATCGTGGGTGAGTTCAT*T*T*A-3' (SEQ ID NO: 38)
　α1-アンチトリプシン欠乏症のPiSの１基変異（PiS：c.863A>T p.Glu288Val）は、α1-アンチトリプシン遺伝子の塩基配列の863位のAがTに変異している１塩基置換である。
　α1-アンチトリプシン遺伝子の二本鎖核酸において、その変異塩基Tを有する側の鎖を第１鎖とし、その相補鎖を第２鎖とした場合、本実施例２で用いたPiS変異型検出用プライマー対のＲプライマーは、第１鎖の変異塩基Tの相補塩基Aを３'末端に有する。Ｒプライマーのその他の塩基配列は、上記第１鎖の上記変異塩基Tに隣接する３’側の塩基配列の相補配列と同じである。
　また、上記PiS変異型検出用プライマー対のＦプライマーは、第２鎖の変異塩基Aの相補塩基Tを３'末端に有する。Ｆプライマーのその他の塩基配列は、上記第２鎖の上記変異塩基Tに隣接する３’側の塩基配列の相補配列と同じである。 Single base mutation of PiS in α1-antitrypsin deficiency (PiS: c.863A>T p.Glu288Val) is a single nucleotide substitution in which A at position 863 of the nucleotide sequence of α1-antitrypsin gene is mutated to T. .
In the double-stranded nucleic acid of the α1-antitrypsin gene, when the strand on the side having the mutated base T is the first strand and the complementary strand is the second strand, The R primer of the primer pair has the complementary base A of the mutated base T of the first strand at the 3' end. The rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the first strand.
In addition, the F primer of the PiS mutation detection primer pair has the complementary base T of the mutated base A of the second strand at the 3' end. The rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
　言い換えれば、ここで用いたPiS変異型検出用プライマー対のＦプライマーは、第１鎖の変異塩基Tを３'末端に有する。Ｆプライマーのその他の塩基配列は、第１鎖の塩基配列の、変異塩基Tより５’側に向かっての塩基配列と同じである。また、PiS変異型検出用プライマー対のＲプライマーは、第１鎖の変異塩基Tの相補塩基Aを３’末端に有する。Ｒプライマーのその他の塩基配列は、第１鎖の変異塩基Tより３’側に向かっての塩基配列の相補配列と同じである。 In other words, the F primer of the PiS mutation detection primer pair used here has the mutated base T of the first strand at the 3' end. The rest of the base sequence of the F primer is the same as the base sequence 5' from the mutated base T in the base sequence of the first strand. In addition, the R primer of the PiS mutation detection primer pair has the complementary base A of the mutated base T of the first strand at the 3' end. The rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence 3' from the mutated base T of the first strand.
２）リアルタイムPCR
　上記1st PCRで得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記１）で得られたＳ化プライマー対（野生型検出用プライマー対、PiS変異型検出用プライマー対）を用いて、実施例１の(２)２)と同様の方法で、PCR反応液を調整し、リアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in the above 1st PCR as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, and using the S-primer pair (for wild-type detection) obtained in 1) above. A primer pair and a PiS mutation detection primer pair) were used to prepare a PCR reaction solution and perform real-time PCR in the same manner as in Example 1 (2) 2).
（４）結果
　得られた増幅曲線を図７に示す。
　図７において、（１）はCOLO201のゲノムDNAを鋳型として用いた結果を、（２）はHepG2のゲノムDNAを鋳型として用いた結果を、（３）はA549のゲノムDNAを鋳型として用いた結果を、（４）はMCF7のゲノムDNAを鋳型として用いた結果をそれぞれ示す。 (4) Results The obtained amplification curve is shown in FIG.
In FIG. 7, (1) is the result of using COLO201 genomic DNA as a template, (2) is the result of using HepG2 genomic DNA as a template, and (3) is the result of using A549 genomic DNA as a template. and (4) show the results using MCF7 genomic DNA as a template, respectively.
　更に、図７において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線ＭはPiS変異型検出用プライマー対を用いた結果をそれぞれ示す。 Furthermore, in FIG. 7, the amplification curve W shows the results using the wild-type detection primer pair, and the amplification curve M shows the results using the PiS mutation type detection primer pair.
　本発明の３塩基がＳ化されたプライマーのプライマー対を用いた検出の結果、COLO201（図７(１)）及びA549（図７(３)）では野生型検出用プライマー対を用いた増幅曲線と変異型検出用プライマー対を用いた増幅曲線が近接していた。このことから、COLO201及びA549は、ヘテロ接合型のPiS変異を持つと判定できた。 As a result of detection using a primer pair of primers in which 3 bases are S of the present invention, COLO201 (Fig. 7 (1)) and A549 (Fig. 7 (3)) Amplification curve using a wild-type detection primer pair and the amplification curves using the primer pair for mutation type detection were close to each other. From this, COLO201 and A549 could be determined to have heterozygous PiS mutations.
　一方、HepG2（図７(２)）及びMCF7（図７(３)）を用いた検出の結果、野生型検出用プライマー対を用いた増幅曲線の方が変異型検出用プライマー対を用いた増幅曲線よりも早く立ち上がっていた（より早期に検出された）。以上のことから、HepG2細胞及びMCF7細胞は、PiS変異を持たない野生型であると判定できた。 On the other hand, as a result of detection using HepG2 (FIG. 7 (2)) and MCF7 (FIG. 7 (3)), the amplification curve using the wild-type detection primer pair is the amplification using the mutation type detection primer pair. It rose earlier than the curve (detected earlier). From the above, the HepG2 cells and MCF7 cells were determined to be wild-type without PiS mutation.
実施例３．α1-アンチトリプシンの変異の検出－２(PiZ)
　実施例３では、本発明のプライマー対を用い、PiZ変異の検出を行った。 Example 3. Mutation detection of α1-antitrypsin-2 (PiZ)
In Example 3, PiZ mutation was detected using the primer pair of the present invention.
（１）ゲノムDNAの調製
　COLO201（ヒト結腸癌細胞、ATCC No. CCL-224）、A549（ヒト肺癌細胞、ATCC No. CCL-185）、HepG2（ヒト肝癌細胞、ATCC No. HB-8065）及びMCF7（ヒト乳癌細胞、ATCC No. HTB-22）の培養液を遠心分離し、上清を除去後、細胞を回収した。QuickGene SP kit DNA tissue（倉敷紡績(株)）を用い、同キットのプロトコールに従って、回収した細胞中のゲノムDNAを抽出した。 (1) Preparation of genomic DNA COLO201 (human colon cancer cells, ATCC No. CCL-224), A549 (human lung cancer cells, ATCC No. CCL-185), HepG2 (human liver cancer cells, ATCC No. HB-8065) and A culture solution of MCF7 (human breast cancer cells, ATCC No. HTB-22) was centrifuged, and after removing the supernatant, the cells were collected. Using QuickGene SP kit DNA tissue (Kurashiki Spinning Co., Ltd.), genomic DNA in the collected cells was extracted according to the protocol of the kit.
（２）1st PCR
　上記（１）で抽出した各細胞のゲノムDNA（n=2）100pgを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (2) 1st PCR
Using 100 pg of genomic DNA (n = 2) of each cell extracted in (1) above as a template, using KOD-Plus-Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, PCR amplification reaction was performed by the following method. gone
１）ゲノムDNAのPCR増幅用プライマー対
　下記のプライマー対を設計し、合成した。
　　Ｆプライマー：5’-GGATCAGCCTTACAACGTGTCTCTGC-3’ （配列番号３９）
　　Ｒプライマー：5’-CAAAGGGTTTGTTGAACTTGACCTCG-3’ （配列番号４０） 1) Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
F primer: 5'-GGATCAGCCTTACAACGTGTCTCTGC-3' (SEQ ID NO: 39)
R primer: 5'-CAAAGGGTTTGTTGAACTTGACCTCG-3' (SEQ ID NO: 40)
２）PCR反応液の調整および1st-PCR
　実施例２の(２)２)及び３)と同様の方法で、PCR反応液の調整、および各細胞由来のゲノムDNAを鋳型として用いたPCR増幅を行い、PCR増幅産物を得た。
　この1st-PCRでは、α1-アンチトリプシン遺伝子のPiZ変異が起きる位置である1096位の塩基を含む領域が増幅される。 2) Preparation of PCR reaction mixture and 1st-PCR
In the same manner as in (2), 2) and 3) of Example 2, the PCR reaction solution was prepared and PCR amplification was performed using genomic DNA derived from each cell as a template to obtain a PCR amplification product.
This 1st-PCR amplifies a region containing base 1096, which is the position where the PiZ mutation of the α1-antitrypsin gene occurs.
（３）リアルタイムPCR (3) Real-time PCR
１）Ｓ化プライマー対
　下記の３個のヌクレオチドがＳ化されたプライマーのプライマー対を設計し、合成した。
　尚、各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 1) S-Primer Pairs The following primer pairs in which three nucleotides were S-substituted were designed and synthesized.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
・PiZ野生型検出用プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCG*A*C*G-3’ （配列番号４９）
　Ｒプライマー：5’-CCCCAGCAGCTTCAGTCCCTTT*C*T*C-3’ （配列番号５０）
・PiZ変異型検出用プライマー対
　Ｆプライマー：5’-CATAAGGCTGTGCTGACCATCG*A*C*A-3’ （配列番号５１）
　Ｒプライマー：5’-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3’ （配列番号５２） ・PiZ wild type detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*G-3' (SEQ ID NO: 49)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*C-3' (SEQ ID NO: 50)
・PiZ mutation detection primer pair F primer: 5'-CATAAGGCTGTGCTGACCATCG*A*C*A-3' (SEQ ID NO: 51)
R primer: 5'-CCCCAGCAGCTTCAGTCCCTTT*C*T*T-3' (SEQ ID NO: 52)
　α1-アンチトリプシン欠乏症のPiZ塩基変異（PiZ：c.1096G>A p.Glu366Lys）は、α1-アンチトリプシン遺伝子の塩基配列の1096位のGがAに変異している１塩基置換である。 The α1-antitrypsin deficiency PiZ nucleotide mutation (PiZ: c.1096G>A p.Glu366Lys) is a single nucleotide substitution in which the G at position 1096 in the nucleotide sequence of the α1-antitrypsin gene is mutated to A.
　α1-アンチトリプシン遺伝子の二本鎖核酸において、その変異塩基Aを有する側の鎖を第１鎖とし、その相補鎖を第２鎖とした場合、本実施例３で用いたPiZ変異型検出用プライマー対のＲプライマーは、第１鎖の変異塩基Aの相補塩基Tを３'末端に有する。Ｒプライマーのその他の塩基配列は、上記第１鎖の上記変異塩基Aに隣接する３’側の塩基配列の相補配列と同じである。
　また、PiZ変異型検出用プライマー対のＦプライマーは、第２鎖の変異塩基Tの相補塩基Aを３'末端に有する。Ｆプライマーのその他の塩基配列は、上記第２鎖の上記変異塩基Tに隣接する３’側の塩基配列の相補配列と同じである。 In the double-stranded nucleic acid of the α1-antitrypsin gene, when the strand on the side having the mutated base A is the first strand and the complementary strand is the second strand, The R primer of the primer pair has the complementary base T of the mutated base A of the first strand at the 3' end. The rest of the base sequence of the R primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base A of the first strand.
In addition, the F primer of the PiZ mutation detection primer pair has the complementary base A of the mutated base T of the second strand at the 3' end. The rest of the base sequence of the F primer is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutated base T of the second strand.
２）リアルタイムPCR
　上記(３)２)で得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記１)で得られたＳ化プライマー対（野生型検出用プライマー対、PiZ変異型検出用プライマー対）を用いて、実施例２の(３)２)と同様の方法で、PCR反応液を調整し、リアルタイムPCRを行った 2) Real-time PCR
Using the PCR amplification product obtained in (3) and (2) above as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, and using the S-primer pair obtained in (1) above (wild Type detection primer pair, PiZ mutation type detection primer pair), a PCR reaction solution was prepared in the same manner as in (3) 2) of Example 2, and real-time PCR was performed.
（４）結果
　得られた増幅曲線を図８に示す。
　図８において、（１）はCOLO201のゲノムDNAを鋳型として用いた結果を、（２）はHepG2のゲノムDNAを鋳型として用いた結果を、（３）はA549のゲノムDNAを鋳型として用いた結果を、（４）はMCF7のゲノムDNAを鋳型として用いた結果をそれぞれ示す。 (4) Results The obtained amplification curves are shown in FIG.
In FIG. 8, (1) is the result of using COLO201 genomic DNA as a template, (2) is the result of using HepG2 genomic DNA as a template, and (3) is the result of using A549 genomic DNA as a template. and (4) show the results using MCF7 genomic DNA as a template, respectively.
　更に、図８において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線Ｍは変異型検出用プライマー対を用いた結果をそれぞれ示す。 Furthermore, in FIG. 8, the amplification curve W shows the results of using the wild-type detection primer pair, and the amplification curve M shows the results of using the mutation-type detection primer pair.
　図８の（１）～（４）から明らかなごとく、本発明の３塩基がＳ化されたプライマーを用いた検出の結果、いずれの細胞も、野生型検出用プライマー対を用いた増幅曲線の方が変異型検出用プライマー対を用いた増幅曲線よりも早く立ち上がっていた（より早期に検出された）。以上のことから、COLO201、HepG2、A549、及びMCF7は、PiZ変異を持たないと判定できた。 As is clear from (1) to (4) in FIG. 8, as a result of detection using the primers having three nucleotides S-sylized according to the present invention, all the cells showed an amplification curve using the wild-type detection primer pair. Amplification curve using the primer pair for mutation type detection rose earlier (detected earlier). From the above, it was determined that COLO201, HepG2, A549, and MCF7 do not have PiZ mutations.
　実施例２及び実施例３の結果から、本発明のＳ化プライマーのプライマー対を用いた変異の検出方法によりヒトのPiS変異及びPiZ変異の遺伝子型を判定できることがわかった。 From the results of Examples 2 and 3, it was found that the genotype of human PiS mutation and PiZ mutation can be determined by the mutation detection method using the primer pair of the S-primer of the present invention.
　PiS変異又はPiZ変異の保因者は、SARS-CoV-2による重症および死亡のリスクが高いことが報告されている（Guy Shapira, et al.,The FASEB Journal. 2020;34:14160-14165、Andrea Vianello, Fausto Braccioni, Arch Bronconeumol Actions, vol.56, No.9, pp. 609-610, 2020）。
　本発明のプライマー対を用い、本発明の方法によりPiS変異やPiZ変異を検出することにより、SARS-CoV-2患者の重症化リスクを予測し、治療方針を考慮する一助となることが期待できる。 Carriers of PiS or PiZ mutations have been reported to be at increased risk of severe illness and death from SARS-CoV-2 (Guy Shapira, et al., The FASEB Journal. 2020;34:14160-14165, Andrea Vianello, Fausto Braccioni, Arch Bronconeumol Actions, vol.56, No.9, pp. 609-610, 2020).
By detecting PiS mutations and PiZ mutations by the method of the present invention using the primer pairs of the present invention, it can be expected to help predict the risk of aggravation of SARS-CoV-2 patients and consider treatment strategies. .
実施例４．PiS １塩基変異検出
（１）試料（コントロールDNA）の調製
　ヒトα1-アンチトリプシン遺伝子の863位の塩基を含む領域の部分塩基配列であり、且つ863位に相当する塩基が変異塩基のTである、塩基配列（配列番号５４）を有するオリゴヌクレオチドを設計し、合成した。このオリゴヌクレオチドを、PiS変異型コントロールDNAとした。
　また、ヒトα1-アンチトリプシン遺伝子の863位の塩基を含む領域の部分塩基配列であり、且つ863位に相当する塩基が野生型のAである塩基配列（配列番号５３）を有するオリゴヌクレオチドを設計し合成した。このオリゴヌクレオチドを、PiS野生型コントロールDNAとした。 Example 4. PiS single nucleotide mutation detection (1) Preparation of sample (control DNA) A partial nucleotide sequence of a region containing the base at position 863 of the human α1-antitrypsin gene, and the base corresponding to position 863 is the mutated base T , an oligonucleotide having the base sequence (SEQ ID NO: 54) was designed and synthesized. This oligonucleotide was used as PiS mutant control DNA.
In addition, an oligonucleotide having a nucleotide sequence (SEQ ID NO: 53), which is a partial nucleotide sequence of a region containing the 863rd nucleotide of the human α1-antitrypsin gene and in which the nucleotide corresponding to the 863rd nucleotide is wild-type A, was designed. and synthesized. This oligonucleotide served as the PiS wild-type control DNA.
（２）Ｓ化プライマー対
　実施例２(３)のリアルタイムPCRで用いたものと同じＳ化プライマー対（PiS野生型検出用プライマー対：配列番号３５－配列番号３６のプライマー対、PiS変異型検出用プライマー対：配列番号３７－配列番号３８のプライマー対）を用いた。 (2) S-primer pair The same S-primer pair as used in real-time PCR in Example 2 (3) (PiS wild-type detection primer pair: SEQ ID NO: 35-SEQ ID NO: 36 primer pair, PiS mutation type detection A primer pair for SEQ ID NO: 37-SEQ ID NO: 38) was used.
（３）リアルタイムPCR
　上記（１）で調整したPiS変異型コントロールDNA又はPiS野生型コントロールDNAを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記（２）のＳ化プライマー対を用いて、以下の方法でリアルタイムPCRを行った。 (3) Real-time PCR
Using the PiS mutant control DNA or PiS wild-type control DNA prepared in (1) above as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, the S-primer of (2) above Using the pairs, real-time PCR was performed by the following method.
１）PCR反応液の調整
　下記の組成のPCR反応液を調整した。
Figure JPOXMLDOC01-appb-I000019
添加したDNAコントロールの量：
　　PiS野生型コントロールDNAのみの場合　　　10pg
　　PiS変異型コントロールDNAのみの場合　　　10pg
　　PiS変異型コントロールDNA及びPiS野生型コントロールの場合　各5pg 1) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000019
Amount of DNA control added:
10 pg for PiS wild-type control DNA only
10 pg for PiS mutant control DNA only
5 pg each for PiS mutant control DNA and PiS wild type control
２）リアルアイムPCR
　上記１)で調製したPCR用反応液20μＬを、96穴反応プレート（Hard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてリアルタイムPCRを行った。即ち、94℃で2分間保温の後、98℃で5秒間、68℃で10秒間の反応を40サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 2) Real-time PCR
20 μL of the reaction solution for PCR prepared in 1) above is placed in wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad), Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（４）結果
　得られた増幅曲線を図９に示す。
　図９(１)は、PiS野生型コントロールDNAのみを試料として用いた結果を示す。この結果は、PiS遺伝子が野生型のモデルである。
　図９（２）は、PiS変異型コントロールDNA及びPiS野生型コントロールDNAの混合液を試料として用いた結果を示す。この結果は、ヘテロ接合型のPiS変異のモデルである。
　図９（３）は、PiS変異型コントロールDNAのみを試料として用いた結果を示す。この結果は、ホモ接合型のPiS変異のモデルである。 (4) Results The obtained amplification curve is shown in FIG.
FIG. 9(1) shows the results using only PiS wild-type control DNA as a sample. This result is a model for the wild-type PiS gene.
FIG. 9(2) shows the results of using a mixed solution of PiS mutant control DNA and PiS wild-type control DNA as a sample. This result is a model for heterozygous PiS mutations.
FIG. 9(3) shows the results using only the PiS mutant control DNA as a sample. This result is a model for homozygous PiS mutations.
　また、図９(１)～(３)において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線ＭはPiS変異型検出用プライマー対を用いた結果をそれぞれ示す。 In addition, in FIGS. 9 (1) to (3), the amplification curve W shows the results using the wild-type detection primer pair, and the amplification curve M shows the results using the PiS mutation type detection primer pair.
　図９(１)から明らかな通り、PiS野生型コントロールDNAのみを試料として用いた場合は、増幅曲線Ｗの方が増幅曲線Ｍよりも先に立ち上がっていた（より早期に検出された）。
　図９(２)から明らかな通り、PiS変異型コントロールDNA及びPiS野生型コントロールDNAの混合液を試料として用いた場合は、増幅曲線Ｗと増幅曲線Ｍが同程度で立ち上がっており、２つの増幅曲線は近接していた（同程度の早さで検出された）。
　図９(３)から明らかな通り、PiS変異型コントロールDNAのみを試料として用いた場合は、増幅曲線Ｗより増幅曲線Ｍの方が先に立ち上がっていた（より早期に検出された）。 As is clear from FIG. 9(1), when only the PiS wild-type control DNA was used as a sample, the amplification curve W rose earlier than the amplification curve M (detected earlier).
As is clear from FIG. 9 (2), when a mixture of PiS mutant control DNA and PiS wild-type control DNA was used as a sample, the amplification curve W and the amplification curve M rose at the same level, and the two amplifications The curves were close (detected as quickly).
As is clear from FIG. 9(3), when only the PiS mutant control DNA was used as a sample, the amplification curve M rose earlier than the amplification curve W (detected earlier).
　以上の結果から明らかな通り、本発明のプライマー対を用いることによって、野生型、ホモ型変異、ヘテロ型変異で増幅曲線の出方が異なることから、得られた増幅曲線を対照として用いて、被検試料の被検対象のα1－アンチトリプシン遺伝子が野生型か、ホモ接合型のPiS変異か、ヘテロ接合型のPiS変異かを区別して判定できることがわかる。 As is clear from the above results, by using the primer pair of the present invention, the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the α1-antitrypsin gene of the subject to be tested in the test sample is wild-type, homozygous PiS mutation, or heterozygous PiS mutation can be discriminated.
実施例５．PiZ １塩基変異検出
（１）試料の調製
　ヒトα1-アンチトリプシン遺伝子の1096位の塩基を含む領域の部分塩基配列であり、且つ1096位に相当する塩基が変異塩基のAである、塩基配列（配列番号５６）を有するオリゴヌクレオチドを設計し、常法により入手した。このオリゴヌクレオチドを、PiZ変異型コントロールDNAとした。
　また、ヒトα1-アンチトリプシン遺伝子の1096位の塩基を含む領域の部分塩基配列であり、且つ1096位に相当する塩基が野生型のGである塩基配列（配列番号５５）を有するオリゴヌクレオチドを設計し、常法により入手した。このオリゴヌクレオチドを、PiZ野生型コントロールDNAとした。 Example 5. PiZ single nucleotide mutation detection (1) sample preparation A nucleotide sequence ( An oligonucleotide having SEQ ID NO: 56) was designed and obtained by standard methods. This oligonucleotide was used as the PiZ mutant control DNA.
In addition, an oligonucleotide having a nucleotide sequence (SEQ ID NO: 55), which is a partial nucleotide sequence of a region containing the 1096th nucleotide of the human α1-antitrypsin gene and in which the nucleotide corresponding to the 1096th nucleotide is a wild-type G, is designed. and obtained by a conventional method. This oligonucleotide served as the PiZ wild-type control DNA.
（２）Ｓ化プライマー対
　実施例３(４)のリアルタイムPCRで用いたものと同じＳ化プライマー対（野生型検出用プライマー対：配列番号４９－配列番号５０のプライマー対、PiZ変異型検出用プライマー対：配列番号５１－配列番号５２のプライマー対）を用いた。 (2) S-primer pair The same S-primer pair as used in real-time PCR in Example 3 (4) (primer pair for wild type detection: primer pair of SEQ ID NO: 49-SEQ ID NO: 50, for detecting PiZ mutation type Primer pair: primer pair of SEQ ID NO:51-SEQ ID NO:52) was used.
（３）リアルタイムPCR
　上記（１）で調整したPiZ変異型コントロールDNA又はPiZ野生型コントロールDNAを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記（２）のＳ化プライマー対を用いて、以下の方法でリアルタイムPCRを行った (3) Real-time PCR
Using the PiZ mutant control DNA or PiZ wild-type control DNA prepared in (1) above as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, the S-primer of (2) above Using the pair, real-time PCR was performed by the following method.
１）PCR反応液の調整
　下記の組成のPCR反応液を調整した。
Figure JPOXMLDOC01-appb-I000020
添加したDNAコントロールの量：
　　PiZ野生型コントロールDNAのみの場合　　　10pg
　　PiZ変異型コントロールDNAのみの場合　　　10pg
　　PiZ変異型コントロールDNA及びPiZ野生型コントロールの場合　　各5pg 1) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000020
Amount of DNA control added:
10 pg for PiZ wild-type control DNA only
10 pg for PiZ mutant control DNA only
5 pg each for PiZ mutant control DNA and PiZ wild type control
２）リアルアイムPCR
　上記１)で調製したPCR用反応液20μＬを、96穴反応プレート（マイクロアンプ・オプチカル・９６ウェル・リアクション・プレート、アプライドバイオシステムズジャパン社製）のウェルに入れ、TaqManTM　PCR 専用サーマルサイクラー・検出器（ABI 7500、アプライドバイオシステムズジャパン社製） を用いてリアルタイムPCRを行った。即ち、94℃で2分間保温の後、98℃で5秒間、68℃で10秒間の反応を40サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 2) Real-time PCR
20 μL of the reaction solution for PCR prepared in 1) above is placed in the wells of a 96-well reaction plate (Microamp Optical 96-well Reaction Plate, Applied Biosystems Japan), and a thermal cycler for TaqMan ™ PCR detection is used. Real-time PCR was performed using a device (ABI 7500, manufactured by Applied Biosystems Japan). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（４）結果
　得られた増幅曲線を図１０に示す。
　図１０(１)は、PiZ野生型コントロールDNAのみを試料として用いた結果を示す。この結果は、PiZ遺伝子が野生型のモデルである。
　図１０（２）は、PiZ変異型コントロールDNA及びPiZ野生型コントロールDNAの混合液を試料として用いた結果を示す。この結果は、ヘテロ接合型のPiZ変異のモデルである。
　図１０（３）は、PiZ変異型コントロールDNAのみを試料として用いた結果を示す。この結果は、のホモ接合型のPiZ変異のモデルである。 (4) Results The obtained amplification curves are shown in FIG.
FIG. 10(1) shows the results using only PiZ wild-type control DNA as a sample. This result is a model for the wild-type PiZ gene.
FIG. 10(2) shows the results of using a mixed solution of PiZ mutant control DNA and PiZ wild-type control DNA as a sample. This result is a model for heterozygous PiZ mutations.
FIG. 10(3) shows the results of using only the PiZ mutant control DNA as a sample. This result is a model for homozygous PiZ mutations.
　また、図１０(１)～(３)において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線ＭはPiZ変異型検出用プライマー対を用いた結果をそれぞれ示す。 In addition, in FIGS. 10 (1) to (3), the amplification curve W shows the results using the wild-type detection primer pair, and the amplification curve M shows the results using the PiZ mutation type detection primer pair.
　図１０(１)から明らかな通り、PiZ野生型コントロールDNAのみを試料として用いた場合は、増幅曲Ｗ線の方が増幅曲線Ｍよりも先に立ち上がっていた（より早期に検出された。）。
　図１０(２)から明らかな通り、PiZ変異型コントロールDNA及びPiZ野生型コントロールDNAの混合液を試料として用いた場合は、増幅曲線Ｗと増幅曲線Ｍが同程度で立ち上がっており、２つの増幅曲線は近接していた（同程度の早さで検出された）。
　図１０(３)から明らかな通り、PiZ変異型コントロールDNAのみを試料として用いた場合は、増幅曲線Ｗより増幅曲線Ｍの方が先に立ち上がっていた（より早期に検出された。）。 As is clear from FIG. 10(1), when only the PiZ wild-type control DNA was used as a sample, the amplification curve W rose earlier than the amplification curve M (detected earlier). .
As is clear from FIG. 10 (2), when a mixture of PiZ mutant control DNA and PiZ wild-type control DNA was used as a sample, the amplification curve W and the amplification curve M rose at about the same level, and the two amplifications The curves were close (detected as quickly).
As is clear from FIG. 10(3), when only the PiZ mutant control DNA was used as a sample, the amplification curve M rose earlier than the amplification curve W (detected earlier).
　以上の結果から明らかな通り、本発明のプライマー対を用いることによって、野生型、ホモ型変異、ヘテロ型変異で増幅曲線の出方が異なることから、得られた増幅曲線を対照として用いて、被検試料の被検対象のα1－アンチトリプシン遺伝子が野生型か、PiZホモ接合型変異か、PiZヘテロ接合型変異かを区別して判定できることがわかる。 As is clear from the above results, by using the primer pair of the present invention, the appearance of the amplification curve differs depending on the wild type, homozygous mutation, and heterozygous mutation. It can be seen that whether the α1-antitrypsin gene of the test sample is a wild type, a PiZ homozygous mutation, or a PiZ heterozygous mutation can be discriminated.
実施例６．ヒト口腔拭い液及び唾液試料を用いたPiS 1塩基変異検出
（１）試料の調整
・ヒト口腔拭い液（ビジコムジャパンより購入）　10μLをチューブに入れ、湯浴中95℃で6分処理した。
・ヒト唾液（日本人）10μLをチューブに入れ、湯浴中95℃で6分処理した。 Example 6. Detection of PiS 1 base mutation using human oral swab and saliva sample (1) Preparation of sample 10 μL of human oral swab (purchased from Busicom Japan) was placed in a tube and treated at 95° C. for 6 minutes in a hot water bath.
・10 μL of human saliva (Japanese) was placed in a tube and treated in a hot water bath at 95°C for 6 minutes.
（２）1st PCR
　上記（１）で調整したヒト口腔拭い液又はヒト唾液夫々４検体を用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (2) 1st PCR
Using four samples each of the human oral swabs and human saliva prepared in (1) above, KOD-Plus-Neo (Toyobo Co., Ltd.) was used as a heat-resistant DNA polymerase, and PCR amplification was performed by the following method.
１）PCR増幅用プライマー対
　実施例２(２)で用いたものと同じ、ゲノムDNAのPCR増幅用プライマー対（配列番号２５－配列番号２６のプライマー対）を用いた。 1) PCR amplification primer pair The same primer pair for PCR amplification of genomic DNA (primer pair of SEQ ID NO: 25-SEQ ID NO: 26) as used in Example 2(2) was used.
２）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 2) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000021
KOD Plus Neo：1U／μL
Figure JPOXMLDOC01-appb-I000021
KOD Plus Neo: 1U/μL
３）PCR増幅
　上記２)で調製したPCR用反応液20μＬを、96穴反応プレート（Hard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製）を用いてリアルタイムPCRを行った。
　PCRは、98℃で20秒間保温の後、98℃で8秒間、68℃で20秒間の反応を３６サイクル繰り返した。 3) PCR amplification 20 μL of the PCR reaction solution prepared in 2) above was added to a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). It was placed in wells and real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad).
PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
（３）リアルタイムＰＣＲ
１）Ｓ化プライマー対
　実施例２(３)のリアルタイムPCRで用いたものと同じＳ化プライマー対（PiS野生型検出用プライマー対：配列番号３５－配列番号３６のプライマー対、PiS変異型検出用プライマー対：配列番号３７－配列番号３８のプライマー対）を用いた。 (3) Real-time PCR
1) S-primer pair The same S-primer pair as used in real-time PCR in Example 2 (3) (PiS wild-type detection primer pair: SEQ ID NO: 35-SEQ ID NO: 36 primer pair, PiS mutation detection Primer pair: primer pair of SEQ ID NO: 37-SEQ ID NO: 38) was used.
２）リアルタイムPCR
　上記1st PCRで得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD FX Neo（東洋紡(株)）を用い、上記１)のＳ化プライマー対を用いて、以下の方法でリアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in the above 1st PCR as a template, using KOD FX Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, and using the S-primer pair of above 1), real-time PCR by the following method. did
ｉ）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 i) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000022
KOD FX Neo：1U/μL
Figure JPOXMLDOC01-appb-I000022
KOD FX Neo: 1U/μL
ii）リアルアイムPCR
　上記１)で調製したPCR用反応液20μＬを、96穴反応プレートHard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてリアルタイムPCRを行った。即ち、94℃で2分間保温の後、98℃で5秒間、68℃で10秒間の反応を40サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 ii) real-time PCR
20 μL of the reaction solution for PCR prepared in 1) above is placed in the wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad), and real-time Real-time PCR was performed using a PCR device (CFX-96, Bio-Rad). That is, after incubation at 94°C for 2 minutes, the reaction was repeated 40 times at 98°C for 5 seconds and 68°C for 10 seconds. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（４）結果
　得られた増幅曲線を図１１及び図１２に示す。
　図１１は、口腔拭い液を試料として用いた場合の結果を示す。図１１において、(１)～(４)は、４検体の結果をそれぞれ示す。
　図１２は、唾液を試料として用いた場合の結果を示す。図１２において、(１)～(４)は、４検体の結果をそれぞれ示す。 (4) Results The obtained amplification curves are shown in FIGS. 11 and 12. FIG.
FIG. 11 shows the results when mouthwash was used as a sample. In FIG. 11, (1) to (4) show the results of four specimens, respectively.
FIG. 12 shows the results when saliva was used as a sample. In FIG. 12, (1) to (4) show the results of four specimens, respectively.
　また、図１１及び図１２において、ＷはPiS野生型検出用プライマー対を用いて得られた増幅曲線を、ＭはPiS変異型検出用プライマー対を用いて得られた増幅曲線をそれぞれ示す。 In addition, in FIGS. 11 and 12, W indicates the amplification curve obtained using the PiS wild-type detection primer pair, and M indicates the amplification curve obtained using the PiS mutation type detection primer pair.
　図１１より、検体(１)、(２)、(４)の増幅曲線は、増幅曲線Ｗの方が増幅曲線Ｍより先に立ち上がっており（より早期に検出された）、図９(１)と同様の様式であった。よって、検体(１)、(２)、(４)はPiS変異に関しては野生型（野生型）であると判定された。検体(３)の増幅曲線は、増幅曲線Ｗと増幅曲線Ｍが近接して立ち上がっており（同程度の早さで検出された）、図９(２)と同様の様式であった。よって、検体(３)はヘテロ変異型のPiS変異であると判定された。 From FIG. 11, among the amplification curves of the samples (1), (2), and (4), the amplification curve W rises earlier than the amplification curve M (detected earlier), and FIG. It was in the same style as Thus, specimens (1), (2), and (4) were determined to be wild-type (wild-type) with respect to the PiS mutation. In the amplification curve of sample (3), the amplification curve W and the amplification curve M rose close to each other (detected at about the same speed), and had the same pattern as in FIG. 9(2). Therefore, specimen (3) was determined to be a heterozygous PiS mutation.
　また、図１２より、検体(１)～(４)の増幅曲線は、増幅曲線Ｗの方が増幅曲線Ｍより先に立ち上がっており（より早期に検出された）、図９(１)と同様の様式であった。よって、検体(１)～(４)は、PiS変異に関しては野生型（野生型）であると判定された。 In addition, from FIG. 12, among the amplification curves of specimens (1) to (4), the amplification curve W rises earlier than the amplification curve M (detected earlier), similar to FIG. 9 (1). It was the style of Thus, specimens (1)-(4) were determined to be wild-type (wild-type) with respect to the PiS mutation.
実施例７．ヒト口腔拭い液及び唾液試料を用いたPiZ １塩基変異検出
（１）試料の調整
　実施例６で用いたものと同じヒト口腔拭い液及びヒト唾液を用い、同様に熱処理による前処理を行った。 Example 7. PiZ Single Nucleotide Mutation Detection Using Human Oral Wipes and Saliva Samples (1) Preparation of Samples Using the same human oral swabs and human saliva as used in Example 6, pretreatment by heat treatment was performed in the same manner.
（２）1st-PCR
　上記（１）で調整したヒト口腔拭い液又はヒト唾液夫々４検体を用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (2) 1st-PCR
Using four samples each of the human oral swabs and human saliva prepared in (1) above, KOD-Plus-Neo (Toyobo Co., Ltd.) was used as a heat-resistant DNA polymerase, and PCR amplification was performed by the following method.
１)PCR増幅用プライマー対
　実施例３(３)で用いたゲノムDNAのPCR増幅用プライマー対（配列番号３９－配列番号４０のプライマー対）を用いた。 1) PCR Amplification Primer Pair The genomic DNA PCR amplification primer pair (primer pair of SEQ ID NO: 39-SEQ ID NO: 40) used in Example 3(3) was used.
２）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 2) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000023
Figure JPOXMLDOC01-appb-I000023
３）PCR増幅
　上記２)で調製したPCR用反応液20μＬを、９６ 穴反応プレートHard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてPCRを行った。
　PCRは、98℃で20秒間保温の後、98℃で8秒間、68℃で20秒間の反応を３６サイクル繰り返した。 3) PCR amplification 20 μL of the reaction mixture for PCR prepared in 2) above was poured into the wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). , and PCR was performed using a real-time PCR device (CFX-96, Bio-Rad).
PCR was carried out by incubating at 98°C for 20 seconds, followed by 36 cycles of reaction at 98°C for 8 seconds and 68°C for 20 seconds.
（３）リアルタイムＰＣＲ
１）Ｓ化プライマー対
　実施例３(３)のリアルタイムPCRで用いたものと同じＳ化プライマー対（野生型検出用プライマー対：配列番号４９－配列番号５０のプライマー対、PiS変異型検出用プライマー対：配列番号５１－配列番号５２のプライマー対）を用いた。 (3) Real-time PCR
1) S-primer pair The same S-primer pair as used in real-time PCR in Example 3 (3) (primer pair for wild type detection: primer pair of SEQ ID NO: 49-SEQ ID NO: 50, PiS mutation type detection primer pair: primer pair of SEQ ID NO: 51-SEQ ID NO: 52) was used.
２）リアルタイムPCR
　上記1st PCRで得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKODFX Neo （東洋紡(株)）を用い、上記１)のＳ化プライマー対を用いて、以下の方法でリアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in the above 1st PCR as a template, using KODFX Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, and using the S-primer pair of 1) above, real-time PCR is performed by the following method. gone.
ｉ）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 i) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000024
Figure JPOXMLDOC01-appb-I000024
ii）リアルアイムPCR
　上記１)で調製したPCR用反応液20μＬを、96穴反応プレートHard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてリアルタイムPCRを行った。即ち、98℃で20秒保温の後、98℃で5秒間、68℃で5秒間の反応を30サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 ii) real-time PCR
20 μL of the reaction solution for PCR prepared in 1) above is placed in the wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad), and real-time Real-time PCR was performed using a PCR device (CFX-96, Bio-Rad). That is, after incubating at 98°C for 20 seconds, a reaction of 98°C for 5 seconds and 68°C for 5 seconds was repeated for 30 cycles. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（４）結果
　得られた増幅曲線を図１３及び図１４に示す。
　図１３は、口腔拭い液を試料として用いた場合の結果を示す。図１３において、(１)～(４)は、４検体の結果をそれぞれ示す。 (4) Results The obtained amplification curves are shown in FIGS. 13 and 14. FIG.
FIG. 13 shows the results when a mouthwash was used as a sample. In FIG. 13, (1) to (4) show the results of four specimens, respectively.
　図１４は、唾液を試料として用いた場合の結果を示す。図１４において、(１)～(４)は、４検体の結果をそれぞれ示す。 Fig. 14 shows the results when saliva was used as a sample. In FIG. 14, (1) to (4) show the results of four specimens, respectively.
　また、図１３及び図１４において、ＷはPiZ野生型検出用プライマー対を用いて得られた増幅曲線を、ＭはPiZ変異型検出用プライマー対を用いて得られた増幅曲線をそれぞれ示す。 In addition, in FIGS. 13 and 14, W indicates the amplification curve obtained using the PiZ wild-type detection primer pair, and M indicates the amplification curve obtained using the PiZ mutation type detection primer pair.
　図１３より、検体(１)～(４)の増幅曲線は、増幅曲線Ｗの方が増幅曲線Ｍより先に立ち上がっており（より早期に検出された）、図１０(１)と同様の様式であった。よって、検体(１)～(４)はすべてPiZ変異に関しては野生型であると判定された。 From FIG. 13, among the amplification curves of specimens (1) to (4), the amplification curve W rises earlier than the amplification curve M (detected earlier), and the pattern is the same as in FIG. 10 (1). Met. Thus, specimens (1)-(4) were all determined to be wild-type for the PiZ mutation.
　また、図１４より、検体(１)～(４)の増幅曲線は、増幅曲線Ｗの方が増幅曲線Ｍより先に立ち上がっており（より早期に検出された）、図１０(１)と同様の様式であった。よって、検体(１)～(４)はすべてPiZ変異に関しては野生型であると判定された。 Further, from FIG. 14, among the amplification curves of specimens (1) to (4), the amplification curve W rises earlier than the amplification curve M (detected earlier), similar to FIG. 10 (1). It was the style of Thus, specimens (1)-(4) were all determined to be wild-type for the PiZ mutation.
　更に実施例６及び７の結果から、本発明のプライマー対を用いた検出で、口腔拭い液及び唾液を熱処理した試料を用いて、PiS変異およびPiZ変異の検出を行えることが明らかになった。 Further, from the results of Examples 6 and 7, it was clarified that the detection using the primer pair of the present invention can detect PiS mutations and PiZ mutations using heat-treated samples of oral swabs and saliva.
実施例８．ネコの塩基配列変異の検出
　ネコでは、以下の遺伝子の１塩基置換が知られている。
　　　PKD1 :  c. 9864C>A
　　　PKLR :  c. 693 + 304G> A 
　実施例８では、本発明のプライマー対を用い、ネコのPKD1遺伝子変異及びPKLR遺伝子変異の検出を行った。 Example 8. Detection of Nucleotide Sequence Mutations in Cats In cats, the following single-nucleotide substitutions in genes are known.
PKD1 : c.9864C>A
PKLR: c.693 + 304G > A
In Example 8, the primer pairs of the present invention were used to detect feline PKD1 and PKLR gene mutations.
（１）DNACaptureの調製
　スコティッシュフォールド（ネコ、飼い猫）の、PKLR遺伝子に関して野生型であることが確認されている個体（n=2）、及び変異型であることが確認されている個体（n=2）の唾液を試料として用いた。
　尚、両個体もPKD1遺伝子に関しては野生型であることが確認されている。
　ネコから採取した唾液を綿棒に含ませて、DNACapture（一般社団法人生命科学教育研究所提供）のセルロース膜に塗布し乾燥させた。 (1) Preparation of DNACapture Scottish fold (cat, domestic cat) individuals confirmed to be wild-type (n = 2) and mutants (n = 2) with respect to the PKLR gene =2) saliva was used as a sample.
It has been confirmed that both individuals are wild-type with respect to the PKD1 gene.
A cotton swab was soaked with saliva collected from a cat, applied to a cellulose membrane of DNACapture (provided by Institute for Life Science Education), and dried.
（２）ゲノムDNAの抽出
　上記（１）で得られたDNACaptureを常法によりパンチングし、そのセルロース膜を1.5mLチューブに入れ、0.5% Tween20を含有する10mM Tris-HCl（pH8.0)溶液を50μL添加して90℃、6分間加熱し、セルロース膜からゲノムDNAを抽出した。 (2) Genomic DNA extraction The DNACapture obtained in (1) above was punched by a conventional method, the resulting cellulose membrane was placed in a 1.5 mL tube, and a 10 mM Tris-HCl (pH 8.0) solution containing 0.5% Tween20 was added. 50 μL was added and heated at 90° C. for 6 minutes to extract genomic DNA from the cellulose membrane.
（３）1st PCR
　上記（２）で抽出したネコ由来のゲノムDNAを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (3) 1st PCR
Using the feline-derived genomic DNA extracted in (2) above as a template and using KOD-Plus-Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, PCR amplification was performed by the following method.
１）プライマー対
・ゲノムDNAのPCR増幅用プライマー対（PKD1変異検出用）
　下記のプライマー対を設計し、合成した。
　　Fプライマー：5’-CTTTTTTGACAAGCATCTCTGGCTCTC-3’ （配列番号５７）
　　Rプライマー：5’-CTGTGTGGCAGGAGAGGAGAGAGAC-3’ （配列番号５８）
・ゲノムDNAのPCR増幅用プライマー対（PKLR変異検出用）
　下記のプライマー対を設計し、合成した。
　　Fプライマー：5’-CCAAGGTGGACAGGCAATAGGACAC-3’ （配列番号５９）
　　Rプライマー：5’-GCAAACACGATGTCTACCTCATGCTC-3’ （配列番号６０） 1) Primer pair ・Primer pair for PCR amplification of genomic DNA (for PKD1 mutation detection)
The following primer pairs were designed and synthesized.
F primer: 5'-CTTTTTGACAAGCATCTCTGGCTCTC-3' (SEQ ID NO: 57)
R primer: 5'-CTGTGTGGCAGGAGAGGAGAGAGAC-3' (SEQ ID NO: 58)
・Primer pair for PCR amplification of genomic DNA (for PKLR mutation detection)
The following primer pairs were designed and synthesized.
F primer: 5'-CCAAGGTGGACAGGCAATAGGACAC-3' (SEQ ID NO: 59)
R primer: 5'-GCAAACACGATGTCTACCTCATGCTC-3' (SEQ ID NO: 60)
２）PCR反応液の調整および1st-PCR
　実施例２の(２)と同様の方法で、試料由来のゲノムDNAを鋳型として用い、PCR反応液の調整およびPCR増幅反応を行い、PCR増幅産物を得た。
　この1st-PCRでは、PKD1遺伝子の9864位の塩基を含む領域が増幅される。
　また、この1st-PCRでは、PKLR遺伝子の693位+304位の塩基を含む領域が増幅される。 2) Preparation of PCR reaction mixture and 1st-PCR
In the same manner as in Example 2 (2), the sample-derived genomic DNA was used as a template to prepare a PCR reaction solution and perform PCR amplification to obtain a PCR amplification product.
In this 1st-PCR, a region containing base 9864 of the PKD1 gene is amplified.
In addition, in this 1st-PCR, a region containing bases at positions 693+304 of the PKLR gene is amplified.
（４）リアルタイムPCR
１）Ｓ化プライマー対
　下記の３個のヌクレオチドがＳ化されたプライマーのプライマー対を設計し、合成した。
　尚、各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 (4) Real-time PCR
1) S-Primer Pairs The following primer pairs in which three nucleotides were S-substituted were designed and synthesized.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
・PKD1野生型検出用プライマー対
　Fプライマー：5’-GTCCAGCGGGCCACCTGT*T*G*C-3’ （配列番号６１）
　Rプライマー: 5’-CAGGAAGAGGCAGACGAGGAGG*A*C*G-3’ （配列番号６２）
・PKD1変異型検出用プライマー対
　Fプライマー：5’-GTCCAGCGGGCCACCTGT*T*G*A-3’ （配列番号６３）
　Rプライマー：5’-CAGGAAGAGGCAGACGAGGAGG*A*C*T-3’ （配列番号６４）
・PKLR野生型検出用プライマー対
　Fプライマー：5’-CCCCGTGCCCCCGCTCC*A*C*G-3’ （配列番号６５）
　Rプライマー: 5’-GTCAGGGGCGAGCCGGGGGCAGA*G*T*C-3’ （配列番号６６）
・PKLR変異型検出用プライマー対
　Fプライマー：5’-CCCCGTGCCCCCGCTCC*A*C*A-3’ （配列番号６７）
　Rプライマー：5’-GTCAGGGGCGAGCCGGGGGCAGA*G*T*T-3’ （配列番号６８） ・PKD1 wild type detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*C-3' (SEQ ID NO: 61)
R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*G-3' (SEQ ID NO: 62)
・PKD1 mutation detection primer pair F primer: 5'-GTCCAGCGGGCCACCTGT*T*G*A-3' (SEQ ID NO: 63)
R primer: 5'-CAGGAAGAGGCAGACGAGGAGG*A*C*T-3' (SEQ ID NO: 64)
・PKLR wild type detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*G-3' (SEQ ID NO: 65)
R primer: 5'-GTCAGGGGCGAGCCGGGGGCAGA*G*T*C-3' (SEQ ID NO: 66)
・PKLR mutation detection primer pair F primer: 5'-CCCCGTGCCCCCGTCCC*A*C*A-3' (SEQ ID NO: 67)
R primer: 5'-GTCAGGGGCGAGCCGGGGGCAGA*G*T*T-3' (SEQ ID NO: 68)
２）リアルタイムPCR
　上記1st PCRで得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記１）で得られたＳ化プライマー対（野生型検出用プライマー対、PiS変異型検出用プライマー対）を用いて、実施例１の(２)２)と同様の方法で、PCR反応液を調整し、リアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in the above 1st PCR as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, and using the S-primer pair (for wild-type detection) obtained in 1) above. A primer pair and a PiS mutation detection primer pair) were used to prepare a PCR reaction solution and perform real-time PCR in the same manner as in Example 1 (2) 2).
（５）結果
　得られた増幅曲線を図１５及び図１６に示す。
　図１５は、PKD1遺伝子及びPKLR遺伝子が野生型であることが確認されているネコ由来のゲノムDNAを鋳型として用いた結果を示す。 (5) Results The obtained amplification curves are shown in FIGS. 15 and 16. FIG.
FIG. 15 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene and PKLR gene have been confirmed to be wild-type.
　図１６は、PKD1遺伝子が野生型で、PKLR遺伝子が変異型であることが確認されているネコ由来のゲノムDNAを鋳型として用いた結果をそれぞれ示す。 Fig. 16 shows the results of using, as a template, genomic DNA derived from cats in which the PKD1 gene was confirmed to be wild-type and the PKLR gene was confirmed to be mutant.
　また、図１５及び図１６において、(１)はPKD1検出用プライマー対を用いた結果を、(２)はPKLR検出用プライマー対を用いた結果を示す。
　また、図１５及び図１６において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線ＭはPKD1変異検出用プライマー対又はPKLR変異検出用プライマー対を用いた結果をそれぞれ示す。 15 and 16, (1) shows the results using the PKD1 detection primer pair, and (2) shows the results using the PKLR detection primer pair.
15 and 16, the amplification curve W shows the results using the wild-type detection primer pair, and the amplification curve M shows the results using the PKD1 mutation detection primer pair or the PKLR mutation detection primer pair.
　PKD1遺伝子及びPKLR遺伝子が野生型であることが確認されているネコ由来のゲノムDNAを鋳型として用いた場合（図１５）、図１５(１)から明らかな通り、PKD1野生型検出用プライマー対を用いた増幅曲線の方がPKD1変異変異型検出用プライマー対を用いた増幅曲線よりも早く立ち上がっていた（より早期に検出された）。 When a genomic DNA derived from a cat in which the PKD1 gene and the PKLR gene have been confirmed to be wild-type is used as a template (Fig. 15), as is clear from Fig. 15 (1), a primer pair for detecting the PKD1 wild-type is used. The amplification curve using the PKD1 mutation type rose earlier than the amplification curve using the primer pair for detecting the PKD1 mutation (detected earlier).
　また、図１５(２)から明らかな通り、PKLR野生型検出用プライマー対を用いた増幅曲線の方がPKLR変異型検出用プライマー対を用いた増幅曲線よりも早く立ち上がっていた（より早期に検出された）。
　以上のことから、被検体のネコはPKD1変異及びPKLR変異を持たない野生型であると判定できた。 In addition, as is clear from FIG. 15 (2), the amplification curve using the PKLR wild-type detection primer pair rose earlier than the amplification curve using the PKLR mutation type detection primer pair (earlier detection was done).
Based on the above, it was determined that the subject cat was a wild-type cat with no PKD1 mutation or PKLR mutation.
　PKD1遺伝子は野生型で、PKLR遺伝子変異を持つことが確認されているネコ由来のゲノムDNAを鋳型として用いた場合（図１６）、図１６(１)から明らかな通り、PKD1野生型検出用プライマー対を用いた増幅曲線の方が、PKD1変異変異型検出用プライマー対を用いた増幅曲線よりも早く立ち上がっていた（より早期に検出された）。
　一方、図１５(２)から明らかな通り、PKLR野生型検出用プライマー対を用いた増幅曲線とPKLR変異変異型検出用プライマー対を用いた増幅曲線は近接していた。
　以上のことから、被検体のネコはPKD1に関しては野生型であるが、PKLR変異を持ち、且つその変異はヘテロ接合型であると判定できた。 The PKD1 gene is wild-type, and when genomic DNA derived from a cat that has been confirmed to have a PKLR gene mutation is used as a template (Fig. 16), as is clear from Fig. 16 (1), a PKD1 wild-type detection primer The amplification curve using the pair rose earlier (detected earlier) than the amplification curve using the primer pair for detecting the PKD1 mutation type.
On the other hand, as is clear from FIG. 15(2), the amplification curve using the PKLR wild-type detection primer pair and the amplification curve using the PKLR mutation type detection primer pair were close to each other.
From the above, it was determined that the subject cat was wild-type with respect to PKD1, but had a PKLR mutation, and that the mutation was heterozygous.
実施例９．イヌの塩基配列変異の検出
　イヌでは、VPS13B遺伝子の4411950位～4411953位のGTTTが欠損している４塩基欠損変異が知られている（g.4411950_4411953delGTTT）。
　実施例１１では、本発明のプライマー対を用い、イヌのVPS13B遺伝子変異変異の検出を行った。 Example 9. Detection of Nucleotide Sequence Mutations in Dogs Dogs are known to have a 4-nucleotide deletion mutation that lacks GTTT at positions 4411950-4411953 of the VPS13B gene (g.4411950_4411953delGTTT).
In Example 11, the primer pair of the present invention was used to detect canine VPS13B gene mutation.
（１）DNACaptureの調製
　チワワ及びシーズ（イヌ、飼い犬）（各n=1）について、VPS13B遺伝子が野生型であることが確認されている個体の唾液を試料として用いた。
　試料を、実施例８（１）と同様の方法でDNACaptureのセルロース膜に塗布し乾燥させた。 (1) Preparation of DNACapture For Chihuahuas and Sheeds (dogs, domestic dogs) (n=1 each), saliva from individuals confirmed to have a wild-type VPS13B gene was used as a sample.
The sample was applied to the cellulose membrane of DNACapture and dried in the same manner as in Example 8(1).
（２）ゲノムDNAの抽出
　上記（１）で得られたDNACaptureを用い、実施例８（２）と同様の方法でゲノムDNAを抽出した。 (2) Extraction of Genomic DNA Using the DNACapture obtained in (1) above, genomic DNA was extracted in the same manner as in Example 8 (2).
（３）1st PCR
　上記（２）で抽出した各細胞のゲノムDNAを鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-Plus-Neo（東洋紡(株)）を用い、以下の方法でPCR増幅反応を行った (3) 1st PCR
Using the genomic DNA of each cell extracted in (2) above as a template and using KOD-Plus-Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, PCR amplification reaction was carried out by the following method.
１）ゲノムDNAのPCR増幅用プライマー対
　下記のプライマー対を設計し、合成した。
　　Fプライマー：5’-CCGTGCTTCTTTGTAGCGTACAAGGAGTAG-3’（配列番号６９）
　　Rプライマー：5’-CAGAGGACAAAGGACTTGTGGTACATCC-3’（配列番号７０） 1) Primer pair for PCR amplification of genomic DNA The following primer pair was designed and synthesized.
F primer: 5'-CCGTGCTTTCTTTGTAGCGTACAAGGAGTAG-3' (SEQ ID NO: 69)
R primer: 5'-CAGAGGACAAAGGACTTGTGGTACATCC-3' (SEQ ID NO: 70)
２）PCR反応液の調整および1st-PCR
　実施例２の(２)と同様の方法で、各試料由来のゲノムDNAを鋳型として用い、PCR反応液の調整およびPCR増幅反応を行い、PCR増幅産物を得た。
　この1st-PCRでは、VPS13B遺伝子の4411950位～4411953位の塩基を含む領域が増幅される。 2) Preparation of PCR reaction mixture and 1st-PCR
In the same manner as in Example 2 (2), genomic DNA derived from each sample was used as a template to prepare a PCR reaction solution and perform PCR amplification to obtain a PCR amplification product.
In this 1st-PCR, a region containing bases 4411950 to 4411953 of the VPS13B gene is amplified.
（４）リアルタイムPCR
１）Ｓ化プライマー対
　下記の３個のヌクレオチドがＳ化されたプライマーのプライマー対を設計し、合成した。
　尚、各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。 (4) Real-time PCR
1) S-Primer Pairs The following primer pairs in which three nucleotides were S-substituted were designed and synthesized.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
・VPS13B野生型検出用プライマー対
　Ｆプライマー：5’-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T*T-3’（配列番号７１）
　Ｒプライマー: 5’-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3’（配列番号７２）
・VPS13B変異型検出用プライマー対
　Ｆプライマー：5’-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3’（配列番号７３）
　Ｒプライマー：5’-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3’（配列番号７４） ・VPS13B wild type detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGCTTG*T*T*T-3' (SEQ ID NO: 71)
R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGATAA*A*C*A-3' (SEQ ID NO: 72)
・ VPS13B mutation detection primer pair F primer: 5'-GCAGTTAATATTGACCCAGTCTTATATAACTGGC*T*T*A-3' (SEQ ID NO: 73)
R primer: 5'-GTCTACTGGTTCGTTTCTGAGGCTGAT*A*A*G-3' (SEQ ID NO: 74)
２）リアルタイムPCR
　上記1st PCRで得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記１）で得られたＳ化プライマー対（野生型検出用プライマー対、PiS変異型検出用プライマー対）を用いて、実施例１の(２)２)と同様の方法で、PCR反応液を調整し、リアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in the above 1st PCR as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a thermostable DNA polymerase, and using the S-primer pair (for wild-type detection) obtained in 1) above. A primer pair and a PiS mutation detection primer pair) were used to prepare a PCR reaction solution and perform real-time PCR in the same manner as in Example 1 (2) 2).
（５）結果
　得られた増幅曲線を図１７に示す。図１７において、増幅曲線Ｗは野生型検出用プライマー対を用いた結果を、増幅曲線ＭはVPS13B変異検出用プライマー対を用いた結果をそれぞれ示す。
　尚、図１７において、チワワ及びシーズ由来の試料を用いた増幅曲線Ｗ及び増幅曲線Ｍは重複したため、それぞれ見かけ上一本になっている。 (5) Results The obtained amplification curve is shown in FIG. In FIG. 17, amplification curve W shows the results using the wild-type detection primer pair, and amplification curve M shows the results using the VPS13B mutation detection primer pair.
In FIG. 17, the amplification curve W and the amplification curve M using samples derived from Chihuahua and seeds are overlapped, so they appear to be one.
　本発明の３塩基がＳ化されたプライマーを用いた検出の結果、VPS13B変異に対して野生型のイヌにおいて、VPS13B野生型検出用プライマー対を用いた核酸増幅反応（Ｗ）では増幅産物が得られたが、VPS13B変異型検出用プライマー対を用いた核酸増幅反応（Ｍ）では増幅産物が得られなかった。このことから、本発明のプライマー対を用いて、対象の遺伝子が野生型であることを判定できることがわかる。 As a result of detection using the primers of the present invention having three bases S-sylized, an amplified product was obtained in the wild-type dog against the VPS13B mutation in the nucleic acid amplification reaction (W) using the VPS13B wild-type detection primer pair. However, no amplified product was obtained in the nucleic acid amplification reaction (M) using the primer pair for detecting the VPS13B mutation. This indicates that the primer pair of the present invention can be used to determine whether the gene of interest is of the wild type.
実施例１０．SARS-CoV-2ウイルスのN501Y及びE484K変異の判定
　SARS-CoV-2ウイルスの以下の変異に着目した。 Example 10. Determination of N501Y and E484K Mutations of SARS-CoV-2 Virus We focused on the following mutations of SARS-CoV-2 virus.
　N501Y変異：イギリス変異株、南アフリカ変異株、ブラジル変異株で確認された変異である。SARS-CoV-2ウイルスの有する塩基配列（GenBank、Accession No. MN908947.3）のA23063Tの１塩基置換である。すなわち、SARS-CoV-2ウイルスのRNAの23063番目の塩基がＡ→Ｔに変異している変異であり、スパイクタンパク質の501番目のアミノ酸のアスパラギンがチロシンに変異している変異である。
　SARS-CoV-2ウイルスのN501Y変異株は、スパイクタンパク質の受容体結合部分のタンパク質の変異を引き起こし、ヒト及びマウスの受容体ACE２との結合親和性を高める。 N501Y mutation: Mutation identified in British, South African, and Brazilian variants. It is a single nucleotide substitution of A23063T in the nucleotide sequence of the SARS-CoV-2 virus (GenBank, Accession No. MN908947.3). That is, it is a mutation in which the 23063rd base of the RNA of the SARS-CoV-2 virus is mutated from A to T, and a mutation in which the 501st amino acid asparagine of the spike protein is mutated to tyrosine.
The N501Y mutant of the SARS-CoV-2 virus causes protein mutations in the receptor-binding portion of the spike protein, resulting in increased binding affinity with the human and mouse receptor ACE2.
　E484K変異：南アフリカ変異株の変異株、及びブラジル変異株の変異株で確認された変異である。SARS-CoV-2ウイルスの有する塩基配列のG23012Aの１塩基置換である。
すなわち、SARS-CoV-2ウイルスのRNAの23012番目の塩基がG→Aに変異している変異であり、スパイクタンパク質の484番目のアミノ酸のグルタミン酸がリシンに変異している変異である。
　SARS-CoV-2ウイルスのE484K変異株は、ワクチン接種により形成された抗体を中和させる可能性がある。
（１）一本鎖RNAの調整
　上記SARS-CoV-2ウイルスの野生型株の塩基配列を持ち、５'末端にT7 RNA Polymeraseのプロモーターの塩基配列を持つDNA、及び上記の各変異株の塩基配列を持ち、５'末端にT7 RNA Polymeraseのプロモーターの塩基配列を持つDNAを合成した。
　合成した各DNAを鋳型として用い、常法であるT7 RNA Polymeraseによる転写反応を行って、それぞれ一本鎖RNAを合成した。 E484K mutation: A mutation identified in the South African variant and the Brazilian variant. It is a single nucleotide substitution of G23012A in the nucleotide sequence of the SARS-CoV-2 virus.
That is, the 23012th base of the SARS-CoV-2 virus RNA is mutated from G to A, and the 484th amino acid glutamic acid of the spike protein is mutated to lysine.
The E484K variant of the SARS-CoV-2 virus may neutralize antibodies generated by vaccination.
(1) Adjustment of single-stranded RNA DNA having the nucleotide sequence of the wild-type strain of the SARS-CoV-2 virus and having the nucleotide sequence of the T7 RNA Polymerase promoter at the 5' end, and the nucleotides of each of the above mutant strains A DNA having a sequence and a base sequence of a T7 RNA Polymerase promoter at the 5' end was synthesized.
Using each of the synthesized DNAs as a template, a transcription reaction was performed using T7 RNA Polymerase, which is a conventional method, to synthesize single-stranded RNAs.
（２）１st PCR
　上記（１）で調整した各一本鎖RNAを鋳型として用い、Luna Universal Probe One-Step RT-qPCR Kit (NEB社製)を用い、以下の方法でRT-PCR増幅反応を行った (2) 1st PCR
Using each single-stranded RNA prepared in (1) above as a template, using Luna Universal Probe One-Step RT-qPCR Kit (manufactured by NEB), RT-PCR amplification reaction was performed by the following method.
１）核酸のPCR増幅用プライマー
　下記のプライマー対を設計し、合成した。
(i)N501Y変異用
　　Fプライマー：5’-CTATCAGGCCGGTAGCACACCTTG-3’ （配列番号７５）
　　Rプライマー：5’-CCACAAACAGTTGCTGGTGCATGTAG-3’ （配列番号７６） 1) Primers for PCR amplification of nucleic acids The following primer pairs were designed and synthesized.
(i) F primer for N501Y mutation: 5'-CTATCAGGCCGGTAGCACACCTTG-3' (SEQ ID NO: 75)
R primer: 5'-CCACAAACAGTTGCTGGTGCATGTAG-3' (SEQ ID NO: 76)
(ii)E484K変異用
　　Fプライマー：5’-CCAGATGATTTTACAGGCTGCGTTATAGC-3’（配列番号７７）
　　Rプライマー：5’-CAAACAGTTGCTGGTGCATGTAGAAGTTC-3’（配列番号７８） (ii) F primer for E484K mutation: 5'-CCAGATGATTTTACAGGCTGCGTTATAGC-3' (SEQ ID NO: 77)
R primer: 5'-CAAACAGTTGCTGGTGCATGTAGAAGTTC-3' (SEQ ID NO: 78)
２）PCR反応液の調整
　下記の組成のPCR反応液を調整した。 2) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000025
Figure JPOXMLDOC01-appb-I000025
３）PCR増幅
　上記ii)で調製したPCR用反応液２０μＬを、９６ 穴反応プレートHard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてRT-PCRを行った。
　PCRは、55℃で10分間保温、95℃で１分間保温の後、95℃で15秒間、68℃で30秒間の反応を28サイクル繰り返した。
　この１st-PCRでは、SARS-CoV-2ウイルス遺伝子の核酸の各変異の変異部分を含む領域が増幅される。また、SARS-CoV-2ウイルスの野生株を用いた場合には、各変異部分に対応する部分を含む領域が増幅される。 3) PCR amplification 20 μL of the reaction solution for PCR prepared in ii) above was poured into the wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad)). , and RT-PCR was performed using a real-time PCR device (CFX-96, Bio-Rad).
PCR was carried out by repeating 28 cycles of incubation at 55°C for 10 minutes, incubation at 95°C for 1 minute, reaction at 95°C for 15 seconds, and reaction at 68°C for 30 seconds.
This 1st-PCR amplifies the region containing the mutated portion of each mutation in the nucleic acid of the SARS-CoV-2 viral gene. In addition, when the wild strain of SARS-CoV-2 virus is used, the region containing the portion corresponding to each mutation portion is amplified.
（３）リアルタイムPCR
１）Ｓ化プライマー対
　下記の３個のヌクレオチドがＳ化されたプライマーのプライマー対を設計し、常法により合成した。
　尚、各プライマーの塩基配列において、＊印はホスホジエステル結合がＳ化されている位置を示す。
(i)N501Y変異検出用
・N501Y野生型検出用プライマー対
　　Fプライマー：5’-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3’ （配列番号７９）
　　Rプライマー：5’-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3’ （配列番号８０）
・N501Y変異型検出用プライマー対
　　Fプライマー：5’-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3’ （配列番号８１）
　　Rプライマー：5’-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3’ （配列番号８２） (3) Real-time PCR
1) S-Primer Pairs A pair of primers in which the following 3 nucleotides were S-converted was designed and synthesized by a conventional method.
In addition, in the nucleotide sequence of each primer, the * mark indicates the position where the phosphodiester bond is S-converted.
(i) N501Y mutation detection/N501Y wild type detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*A-3' (SEQ ID NO: 79)
R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*T-3' (SEQ ID NO: 80)
・N501Y mutation detection primer pair F primer: 5'-CCTTTACAATCATATGGTTTCCAACCCA*C*T*T-3' (SEQ ID NO: 81)
R primer: 5'-CTACTCTGTATGGTTGGTAACCAACACC*A*T*A-3' (SEQ ID NO: 82)
(ii)E484K変異検出用
・E484K野生型検出用プライマー対
　　Fプライマー：5’-CGGTAGCACACCTTGTAATGGTG*T*T*G-3’（配列番号８３）
　　Rプライマー：5’-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*C-3’ （配列番号８４）
・E484K変異型検出用プライマー対
　　Fプライマー：5’-CGGTAGCACACCTTGTAATGGTG*T*T*A-3’（配列番号８５）
　　Rプライマー：5’-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*T-3’ （配列番号８６） (ii) E484K mutation detection/E484K wild type detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*G-3' (SEQ ID NO: 83)
R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*C-3' (SEQ ID NO: 84)
・E484K mutation detection primer pair F primer: 5'-CGGTAGCACACCTTGTAATGGTG*T*T*A-3' (SEQ ID NO: 85)
R primer: 5'-CCATATGATTGTAAAGGAAAGTAACAATTAAAACC*T*T*T-3' (SEQ ID NO: 86)
２）リアルタイムPCR
　上記(２)３)で得られたPCR増幅産物を鋳型として用い、耐熱性DNAポリメラーゼとしてKOD-FX-Neo（東洋紡(株)）を用い、上記５）で得られた各Ｓ化プライマー対を用い、以下の方法で、リアルタイムPCRを行った。 2) Real-time PCR
Using the PCR amplification product obtained in (2) and (3) above as a template, using KOD-FX-Neo (Toyobo Co., Ltd.) as a heat-stable DNA polymerase, each S-primer pair obtained in (5) above was Real-time PCR was performed using the following method.
ｉ）PCR反応液の調整
　下記の組成のPCR反応液を調整した。
Figure JPOXMLDOC01-appb-I000026
i) Preparation of PCR reaction solution A PCR reaction solution having the following composition was prepared.
Figure JPOXMLDOC01-appb-I000026
ii）リアルアイムPCR
　即ち、上記ｉ）で調製したPCR用反応液２０μＬを、９６ 穴反応プレートHard-ShellTMLow-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well、バイオラッド社製）のウェルに入れ、リアルタイムPCR装置（CFX-96、バイオラッド社製） を用いてリアルタイムPCRを行った。即ち、94℃で２分間保温の後、98℃で５秒間、68℃で10秒間の反応を40サイクル繰り返した。そして、EvaGreenTM由来の蛍光強度を測定した。 ii) real-time PCR
That is, 20 μL of the PCR reaction solution prepared in i) above was placed in the wells of a 96-well reaction plate (Hard-Shell ™ Low-Profile, Thin-Wall, Skirted 96-Well PCR Plates, White Well, Bio-Rad). Real-time PCR was performed using a real-time PCR device (CFX-96, Bio-Rad). That is, after incubating at 94°C for 2 minutes, reaction at 98°C for 5 seconds and at 68°C for 10 seconds was repeated for 40 cycles. Then, fluorescence intensity derived from EvaGreen ™ was measured.
（４）結果
　得られた増幅曲線を図１８～図１９に示す。
　図１８は、N501Y変異を検出した結果である。
　図１９は、E484K変異を検出した結果である。 (4) Results The amplification curves obtained are shown in FIGS.
FIG. 18 shows the results of detecting the N501Y mutation.
FIG. 19 shows the results of detecting the E484K mutation.
　また、図１８及び図１９において、（１）SARS-CoV-2ウイルスの野生株の塩基配列を持つDNAを用いて得られた結果を、（２）はSARS-CoV-2ウイルスのN501Y変異又はE484K変異の塩基配列を持つDNAを用いて得られた結果をそれぞれ示す。 In addition, in FIGS. 18 and 19, (1) the results obtained using DNA having the base sequence of the wild strain of the SARS-CoV-2 virus, (2) the N501Y mutation of the SARS-CoV-2 virus or Results obtained using DNA having the base sequence of the E484K mutation are shown respectively.
　また、図１８及び図１９において、Ｗは野生型検出用プライマー対を用いた結果を、Ｍは変異型検出用プライマー対を用いて得られた結果をそれぞれ示す。 In addition, in FIGS. 18 and 19, W indicates the results obtained using the wild-type detection primer pair, and M indicates the results obtained using the mutation-type detection primer pair.
　図１８（１）及び図１９（１）から明らかなごとく、SARS-CoV-2ウイルスの野生型株の塩基配列を持つDNAを試料とした場合は、N501Y野生型検出用プライマー対を用いて得られた増幅曲線は、N501Y変異型検出用プライマー対を用いて得られた増幅曲線より早期に検出され（図１８(１)）、同様にE484K野生型検出用プライマー対を用いて得られた増幅曲線は、E484K変異型検出用プライマー対を用いて得られた増幅曲線より早期に検出された（図１９(２)）。 As is clear from Figures 18 (1) and 19 (1), when DNA having the base sequence of the wild-type strain of SARS-CoV-2 virus was used as a sample, the N501Y wild-type detection primer pair was used. The resulting amplification curve was detected earlier than the amplification curve obtained using the N501Y mutant detection primer pair (FIG. 18 (1)), and similarly the amplification obtained using the E484K wild type detection primer pair. The curve was detected earlier than the amplification curve obtained using the E484K mutation detection primer pair (Fig. 19(2)).
　一方、図１８（２）及び図１９（２）から明らかなごとく、SARS-CoV-2ウイルスのN501Y変異株又はE484K変異株の塩基配列を持つDNAを試料とした場合は、N501Y野生型検出用プライマー対を用いて得られた増幅曲線は、N501Y変異型検出用プライマー対を用いて得られた増幅曲線より遅くに検出され（図１８(２)）、同様にE484K野生型検出用プライマー対を用いて得られた増幅曲線は、E484K変異型検出用プライマー対を用いて得られた増幅曲線より遅くに検出された（図１９(２)）。 On the other hand, as is clear from FIGS. 18 (2) and 19 (2), when DNA having the nucleotide sequence of the N501Y mutant or E484K mutant of the SARS-CoV-2 virus is used as a sample, N501Y wild type detection The amplification curve obtained using the primer pair was detected later than the amplification curve obtained using the N501Y mutant detection primer pair (Fig. 18 (2)). The amplification curve obtained using E484K was detected later than the amplification curve obtained using the primer pair for detecting the E484K mutation (FIG. 19(2)).
　以上のことから、本発明のプライマー対を用いてことからSARS-CoV-2のN501Y及びE484K変異を検出できることが示された。 From the above, it was shown that the N501Y and E484K mutations of SARS-CoV-2 can be detected using the primer pair of the present invention.

Claims (15)

  1. ３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がホスホロチオエート化（Ｓ化）されているフォワードプライマーと、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーとのプライマー対。 A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is phosphorothioated (S), and a forward primer from the 3' end A primer pair with a reverse primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base toward the 5' side is S-converted.
  2. プライマー対が下記Ｍ又はＷである、請求項１に記載のプライマー対
    　プライマー対Ｍ：
    変異を有する二本鎖核酸の一方の鎖（第２鎖）にアニールするプライマーであり、前記変異を含む領域にアニールし、前記第２鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
    前記変異を有する二本鎖核酸のもう一方の鎖（第１鎖）にアニールするプライマーであり、前記変異を含む領域にアニールし、前記第１鎖の変異配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対、
    　プライマー対Ｗ：
    野生型の二本鎖核酸の一方の鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異に対応する前記野生型の第２鎖中の塩基配列を含む領域にアニールし、前記変異型の第２鎖の前記変異に対応する前記野生型の第２鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
    野生型の二本鎖核酸のもう一方の鎖（野生型の第１鎖）にアニールするプライマーであり、前記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異に対応する前記野生型の第１鎖中の塩基配列を含む領域にアニールし、前記変異型の第１鎖の前記変異に対応する前記野生型の第１鎖中の塩基配列の相補配列を３’末端側に有するものであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。     The primer pair according to claim 1, wherein the primer pair is M or W below Primer pair M:
    A primer that anneals to one strand (second strand) of a double-stranded nucleic acid having a mutation, anneals to a region containing the mutation, and has a sequence complementary to the mutated sequence of the second strand on the 3′ end side. A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the second base from the 3' end toward the 5' side is S-formed,
    A primer that anneals to the other strand (first strand) of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, and anneals the complementary sequence of the mutation sequence of the first strand to the 3' end side A primer pair of a reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed,
    Primer pair W:
    A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the nucleotide sequence in the wild-type second strand, and placing a complementary sequence of the nucleotide sequence in the wild-type second strand corresponding to the mutation in the mutant-type second strand on the 3′ end side A forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-formed,
    A primer that anneals to the other strand of the wild-type double-stranded nucleic acid (wild-type first strand), and for mutation of the first strand of the mutant double-stranded nucleic acid (mutant-type first strand) Annealing to a region containing the corresponding nucleotide sequence in the wild-type first strand, and 3′ the complementary sequence of the nucleotide sequence in the wild-type first strand corresponding to the mutation in the mutant first strand A reverse primer having a phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side, which has a terminal side and is S-ified. primer pair.
  3. 　前記プライマー対Ｍに係る塩基配列の変異が塩基の置換又は挿入であり、前記プライマー対Ｍが下記のものである、請求項２に記載のプライマー対：
    　前記変異を有する二本鎖核酸の第２鎖にアニールするプライマーであり、前記変異を含む領域にアニールし、前記第２鎖の変異配列の相補配列を３’末端側に有し、その他の塩基配列は前記第２鎖の前記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
    　前記変異を有する二本鎖核酸の第１鎖にアニールするプライマーであり、前記変異を含む領域にアニールし、前記第１鎖の変異配列の相補配列を３’末端側に有し、その他の塩基配列は前記第１鎖の前記変異に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。     The primer pair according to claim 2, wherein the mutation in the base sequence of the primer pair M is a base substitution or insertion, and the primer pair M is:
    A primer that anneals to the second strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the second strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the second strand, and has 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side. a forward primer in which the phosphodiester bond on the 3′ side of the nucleotide is S-converted;
    A primer that anneals to the first strand of the double-stranded nucleic acid having the mutation, anneals to the region containing the mutation, has a sequence complementary to the mutated sequence of the first strand on the 3′ end side, and other bases. The sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the mutation of the first strand, and has 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side. A primer pair of reverse primers in which the phosphodiester bond on the 3' side of the nucleotide is S-converted.
  4. 　前記プライマー対Ｍに係る塩基配列の変異が塩基の欠損であり、前記プライマー対Ｍが下記のものである、請求項２に記載のプライマー対：
    　前記欠損を有する二本鎖核酸の第２鎖にアニールするプライマーであり、前記欠損を含む領域にアニールし、前記第２鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端に有し、その他の塩基配列は前記第２鎖の前記欠損に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
    　前記欠損を有する二本鎖核酸の第１鎖にアニールするプライマーであり、前記欠損を含む領域にアニールし、前記第１鎖の欠損に隣接する５’側の塩基配列の相補配列を３’末端に有し、その他の塩基配列は前記第１鎖の前記欠損に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。     The primer pair according to claim 2, wherein the mutation in the nucleotide sequence of the primer pair M is a deletion of a base, and the primer pair M is:
    A primer that anneals to the second strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and anneals to the 3' end of the complementary sequence of the 5' base sequence adjacent to the deletion of the second strand. and the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the second strand, and a forward primer in which the phosphodiester bond on the 3′ side of consecutive 1 to 4 nucleotides is S-formed;
    A primer that anneals to the first strand of the double-stranded nucleic acid having the deletion, anneals to the region containing the deletion, and anneals to the 3' end of the complementary sequence of the 5' base sequence adjacent to the deletion of the first strand. and the other base sequence is the same as the complementary sequence of the base sequence on the 3' side adjacent to the deletion of the first strand, and from the second base from the 3' end to the 5' side A pair of reverse primers in which the phosphodiester bond on the 3' side of consecutive 1 to 4 nucleotides is S-converted.
  5. 　前記プライマー対Ｗが下記のものである、請求項２に記載のプライマー対：
    　野生型の二本鎖核酸の一方の鎖（野生型の第２鎖）にアニールするプライマーであり、変異型の二本鎖核酸の第２鎖（変異型の第２鎖）の変異に対応する前記野生型の第２鎖中の塩基配列を含む領域にアニールし、前記変異型の第２鎖の前記変異に対応する前記野生型の第２鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は前記変異型の第２鎖の前記変異に対応する前記野生型の第２鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているフォワードプライマーと、
    　前記野生型の二本鎖核酸のもう一方の鎖（野生型の第１鎖）にアニールするプライマーであり、前記変異型の二本鎖核酸の第１鎖（変異型の第１鎖）の変異に対応する前記野生型の第１鎖中の塩基配列を含む領域にアニールし、前記変異型の第１鎖の前記変異に対応する前記野生型の第１鎖の塩基配列の相補配列を３’末端側に有し、その他の塩基配列は前記変異型の第１鎖の前記変異に対応する前記野生型の第１鎖の塩基配列に隣接する３’側の塩基配列の相補配列と同じであって、３'末端から２番目の塩基から５’側に向けての連続する１～４個のヌクレオチドの３'側のホスホジエステル結合がＳ化されているリバースプライマーのプライマー対。     3. The primer pair of claim 2, wherein said primer pair W is:
    A primer that anneals to one strand of a wild-type double-stranded nucleic acid (wild-type second strand) and corresponds to a mutation in the second strand of a mutant-type double-stranded nucleic acid (mutant-type second strand) Annealing to a region containing the nucleotide sequence in the wild-type second strand, and placing a complementary sequence of the nucleotide sequence of the wild-type second strand corresponding to the mutation of the mutant second strand on the 3' end side and the other nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the nucleotide sequence of the wild-type second strand corresponding to the mutation of the mutant second strand, a forward primer in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 'end toward the 5' side is S-formed;
    A primer that anneals to the other strand of the wild-type double-stranded nucleic acid (wild-type first strand), and mutation of the first strand of the mutant double-stranded nucleic acid (mutant-type first strand) and annealed to a region containing the nucleotide sequence in the wild-type first strand corresponding to the 3' complementary sequence of the nucleotide sequence of the wild-type first strand corresponding to the mutation in the mutant first strand The rest of the nucleotide sequence is the same as the complementary sequence of the 3′-side nucleotide sequence adjacent to the wild-type first strand nucleotide sequence corresponding to the mutation of the mutant first strand. A pair of reverse primers in which the phosphodiester bond on the 3' side of 1 to 4 consecutive nucleotides from the 2nd base from the 3' end toward the 5' side is S-converted.
  6. プライマーの３'末端から２番目の塩基から５’側に向けての連続する３個のヌクレオチドの３'側のホスホジエステル結合がＳ化されている、請求項１に記載のプライマー対。 2. The primer pair according to claim 1, wherein the phosphodiester bond on the 3' side of three consecutive nucleotides from the second base from the 3' end of the primer toward the 5' side is S-converted.
  7. 前記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、請求項２に記載のプライマー対。 3. A primer pair according to claim 2, wherein said mutation is a PiS mutation or a PiZ mutation in the α1-antitrypsin gene.
  8. 被検試料の核酸を鋳型として用い、請求項１又は請求項２に記載のプライマー対を用いた核酸増幅反応を行って反応産物を検出し、得られた検出結果をもとに、前記核酸の塩基配列の変異を判定する、塩基配列の変異の判定方法。 Using the nucleic acid of the test sample as a template, a nucleic acid amplification reaction is performed using the primer pair according to claim 1 or claim 2 to detect the reaction product, and based on the obtained detection result, the nucleic acid is A method for determining mutation in a base sequence, which determines mutation in a base sequence.
  9. 請求項２に記載のプライマー対を用い、核酸増幅反応を行って反応産物を検出し得られた検出結果をもとに前記核酸の塩基配列の変異を判定する方法が下記の工程を含む、請求項８に記載の判定方法：
    　（１）被検試料の核酸を鋳型として用い、請求項２に記載のプライマー対Ｍを用いた核酸増幅反応を行い、反応産物を検出する工程、
    　（２）前記(１)の工程で用いたものと同じ被検試料の核酸を鋳型として用い、請求項２に記載のプライマー対Ｗを用いた核酸増幅反応を行い、反応産物を検出する工程、
    　（３）前記(１)及び(２)の工程で得られた検出結果をもとに、被検試料の核酸の塩基配列の変異を判定する工程。     A method for determining a mutation in the base sequence of the nucleic acid based on the detection results obtained by performing a nucleic acid amplification reaction using the primer pair according to claim 2 and detecting the reaction product includes the following steps. The determination method according to Item 8:
    (1) using the nucleic acid of the test sample as a template, performing a nucleic acid amplification reaction using the primer pair M according to claim 2, and detecting the reaction product;
    (2) a step of performing a nucleic acid amplification reaction using the primer pair W according to claim 2, using the same nucleic acid of the test sample as that used in the step (1) as a template, and detecting the reaction product;
    (3) A step of judging a mutation in the base sequence of the nucleic acid of the test sample based on the detection results obtained in the steps (1) and (2).
  10. 被検試料の核酸を鋳型として用いた核酸増幅反応を行い、塩基配列の変異が存在し得る領域を増幅し、得られた増幅産物を鋳型として用いる、請求項８に記載の判定方法。 9. The determination method according to claim 8, wherein a nucleic acid amplification reaction is performed using the nucleic acid of the test sample as a template, a region in which nucleotide sequence mutation may exist is amplified, and the resulting amplification product is used as a template.
  11. 前記塩基配列の変異の判定が、被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かを判定すること、又は野生型か変異型かを判定することである、請求項８に記載の判定方法。 Determination of the mutation in the base sequence is to determine whether the base sequence of the nucleic acid in the test sample is a wild type, homozygous mutation, or heterozygous mutation, or to determine whether it is a wild type or a mutant type. , the determination method according to claim 8.
  12. 前記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、請求項８に記載の判定方法。 The determination method according to claim 8, wherein the mutation is PiS mutation or PiZ mutation in the α1-antitrypsin gene.
  13. 請求項１に記載のプライマー対を含む、塩基配列の変異判定用キット。 A nucleotide sequence mutation determination kit comprising the primer pair according to claim 1 .
  14. 被検試料の核酸の塩基配列が野生型かホモ接合型変異か若しくはヘテロ接合型変異かの判定用、又は野生型か変異型かの判定用である、請求項１３に記載のキット。 14. The kit according to claim 13, which is used for determining whether the base sequence of the nucleic acid of the test sample is wild-type, homozygous mutation, or heterozygous mutation, or for determining whether it is wild-type or mutant.
  15. 前記変異がα1-アンチトリプシン遺伝子のPiS変異又はPiZ変異である、請求項１４に記載のキット。 15. The kit of claim 14, wherein said mutation is a PiS mutation or a PiZ mutation in the α1-antitrypsin gene.
PCT/JP2022/012939 2021-03-26 2022-03-22 Primer pair, method for determining base sequence variation, and kit for determining variation in base sequence WO2022202728A1 (en)

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