CN117025730A - Novel RNase H-dependent isothermal amplification method and application thereof - Google Patents
Novel RNase H-dependent isothermal amplification method and application thereof Download PDFInfo
- Publication number
- CN117025730A CN117025730A CN202311005986.7A CN202311005986A CN117025730A CN 117025730 A CN117025730 A CN 117025730A CN 202311005986 A CN202311005986 A CN 202311005986A CN 117025730 A CN117025730 A CN 117025730A
- Authority
- CN
- China
- Prior art keywords
- rnase
- primers
- isothermal amplification
- base segment
- amplification method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102100034343 Integrase Human genes 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 61
- 101710203526 Integrase Proteins 0.000 title claims abstract description 51
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 48
- 230000001419 dependent effect Effects 0.000 title claims abstract description 36
- 108020004414 DNA Proteins 0.000 claims abstract description 98
- 230000003321 amplification Effects 0.000 claims abstract description 96
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 96
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 76
- 102000053602 DNA Human genes 0.000 claims abstract description 43
- 239000012634 fragment Substances 0.000 claims abstract description 30
- 108091028664 Ribonucleotide Proteins 0.000 claims abstract description 25
- 239000002336 ribonucleotide Substances 0.000 claims abstract description 25
- 125000002652 ribonucleotide group Chemical group 0.000 claims abstract description 25
- 239000005547 deoxyribonucleotide Substances 0.000 claims abstract description 20
- 125000002637 deoxyribonucleotide group Chemical group 0.000 claims abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 20
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 19
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 19
- 108010064245 urinary gonadotropin fragment Proteins 0.000 claims abstract description 10
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 54
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 54
- 230000000295 complement effect Effects 0.000 claims description 39
- 238000006073 displacement reaction Methods 0.000 claims description 32
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 27
- 239000003112 inhibitor Substances 0.000 claims description 25
- 239000007850 fluorescent dye Substances 0.000 claims description 21
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 18
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 18
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 18
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 18
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 18
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 18
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 18
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108010083644 Ribonucleases Proteins 0.000 claims description 8
- 102000006382 Ribonucleases Human genes 0.000 claims description 8
- 238000010839 reverse transcription Methods 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 6
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 4
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 claims description 4
- 241000211181 Manta Species 0.000 claims description 3
- NFGXHKASABOEEW-LDRANXPESA-N methoprene Chemical compound COC(C)(C)CCCC(C)C\C=C\C(\C)=C\C(=O)OC(C)C NFGXHKASABOEEW-LDRANXPESA-N 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 claims description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 239000013615 primer Substances 0.000 description 86
- 238000007397 LAMP assay Methods 0.000 description 20
- 238000001514 detection method Methods 0.000 description 17
- 108020004682 Single-Stranded DNA Proteins 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 9
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 3
- 241000713869 Moloney murine leukemia virus Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 206010014599 encephalitis Diseases 0.000 description 3
- 238000013401 experimental design Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 101150013191 E gene Proteins 0.000 description 2
- 101710147059 Nicking endonuclease Proteins 0.000 description 2
- 241001112090 Pseudovirus Species 0.000 description 2
- 108091028733 RNTP Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 102100025290 Ribonuclease H1 Human genes 0.000 description 2
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 108010052833 ribonuclease HI Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 241001678559 COVID-19 virus Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000710842 Japanese encephalitis virus Species 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 239000013616 RNA primer Substances 0.000 description 1
- 101710162453 Replication factor A Proteins 0.000 description 1
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 1
- 101710176276 SSB protein Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a novel RNase H-dependent isothermal amplification method and application thereof. Adding RNase H enzyme or reverse transcriptase with RNase H activity into an amplification system, wherein the adopted primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1 to 10 ribonucleotides, and the number of the ribonucleotides is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid. The invention utilizes the characteristic of RNaseH specific degradation and RNA hybrid chain combined with DNA to open double-chain gaps and accelerate the isothermal amplification process, thereby greatly improving the reaction rate, the specificity and the sensitivity and having very wide application scenes.
Description
Technical Field
The invention relates to the technical field of nucleic acid molecule detection, in particular to a novel RNase H-dependent isothermal amplification method and application thereof.
Background
In recent years, with the rapid development of molecular biology techniques, diagnostic methods based on nucleic acid detection have been established in large numbers and widely used in laboratory detection of diseases, and isothermal amplification techniques have emerged in this context. Compared with other nucleic acid amplification technologies, isothermal amplification has the advantages of rapidness, high efficiency and specificity, and does not need special equipment.
The main isothermal amplification technologies at present are as follows: loop-mediated isothermal amplification (LAMP), nicking endonuclease isothermal amplification (NEAR), nucleic acid sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), melting enzyme amplification (HDA) and Recombinase Polymerase Amplification (RPA), which are each characterized, also determine their use in disease detection.
The principle of LMAP is based on that when DNA is in a dynamic equilibrium state at about 65 ℃, any one primer performs base pairing extension to the complementary part of double-stranded DNA, the other strand is dissociated into single strands, on the premise that 6 specific regions of a target gene are identified by using 4 different specific primers, under the action of strand displacement DNA polymerase, the 3' -end of an outer primer section is taken as an origin to pair with a template DNA complementary sequence, and strand displacement DNA synthesis is started.
LAMP advantage: high amplification efficiency and short reaction time. Disadvantages: the primer has very high requirement, is not easy to design, rejects amplified products, increases non-characteristic amplification, is easy to pollute and has poor sensitivity.
NEAR is a strand displacement amplification technique, and its principle is that at the gap formed by endonuclease nicking, dNTPs are polymerized and extended from the 3' end of the gap by the action of polymerase to displace allelic DNA strand, thus forming a new complete DNA sequence containing nicking enzyme recognition site. This double strand is again cleaved by nicking endonuclease recognition, and the "polymerization-nicking" cycle is started, producing a large number of displaced DNA single strands, resulting in exponential amplification.
NEAR advantage: the reaction speed is high, the sensitivity is high, and the application is wide; disadvantages: the raw materials are expensive.
NASBA is a new technology developed on the basis of PCR, which is a continuous, isothermal, enzyme reaction-based nucleic acid amplification technology guided by 1 pair of primers with T7 promoter sequence, the reaction is carried out at 41℃and the template RNA can be amplified for about 10 hours or so 9 Multiple of ratio ofThe conventional PCR method is 1000 times higher, and no special instrument is needed.
NASBA sensitivity is very high, relative specificity is poor, and the reaction system is not easy to build.
HDA was a novel isothermal amplification technique for nucleic acids invented by NEB corporation in the united states in 2004. The technology simulates the natural process of in vivo DNA replication, unwinds DNA double chains at constant temperature by using helicase, uses the DNA single chain binding protein to stabilize the unwound single chains as a primer to provide a template, synthesizes complementary double chains under the action of DNA polymerase, and continuously repeats the cyclic amplification process to finally realize the exponential growth of a target sequence.
The sensitivity of this technology is the core limiting its widespread use.
RPA is a novel nucleic acid isothermal amplification technology, and can realize rapid detection of a target to be detected within 10-30 min at 37-42 ℃. The recombinase can be tightly combined with the primer DNA at the constant temperature of 37-42 ℃ to form an enzyme and primer aggregate, when the primer searches the sequence completely complementary to the primer on the template DNA, the template DNA is melted with the help of a single-stranded DNA binding protein (single strand dDNAbinding, SSB), and a new DNA complementary strand is formed under the action of DNA polymerase. The reaction product of the reciprocating cycle grows exponentially.
The RPA has the following characteristics:
1) The speed is high: usually 5 to 10 minutes, the amplification of the nucleic acid template can be accomplished.
2) The reaction temperature is low: the reaction can be carried out at 37 ℃ or even at normal temperature.
RPA disadvantage:
1) The specificity is poor: the RPA primer is 28-35 bp, and nonspecific amplification is easy to generate.
2) The sensitivity is low: because of the very large number of nonspecific amplifications, the target amplification efficiency is low and the sensitivity is poor.
3) The optimal design difficulty is high: in particular, the real-time fluorescence method RPA of the probe method adopts EXOIII and NFO enzyme to selectively identify the TNT sequence. Thus, the probe is 40 to 55 bases and requires a "TNT" specific sequence, where N is any base other than A, and a continuous "TTT" structure is generally selected. Labeling fluorescent groups (F) and quenching groups (Q) at two ends T respectively; common fluorophores are: FAM, HEX, VIC, ROX, cy3, cy5, NED, etc.; common quenching groups: BHQ1, BHQ2, BHQ3, etc.
For the reasons mentioned above, the clinical use of RPA is greatly limited.
RNase H specifically degrades only double stranded RNA: RNA in DNA hybrids, therefore, is commonly used as a laboratory reagent in molecular biology. RNase H1 is commonly used to disrupt RNA templates after cDNA synthesis by reverse transcription, and it can also be used to cleave specific RNA sequences in the presence of short complementary fragments of DNA. RNase H2 can be used to degrade the RNA primer component of the okazaki fragment or to introduce single stranded cleavage at a site containing ribonucleotides.
RNase H enzyme has been widely used in PCR (rh-PCR), LAMP (fluorescent) amplification or other isothermal amplification (such as Shanghai-Haoren biological SAT technology), but its main function is to hydrolyze probes to generate fluorescence, for example, 4 ribonucleic acid (rUTP) -modified TaqMan probes are selected in patent CN201910284681.1, and RNase H1 is used for selective cleavage instead of playing an important role in the amplification process. The invention aims to provide a novel isothermal amplification technology which utilizes RNase H enzyme to improve reaction rate, specificity and sensitivity.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a novel RNase H-dependent isothermal amplification method and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the first aspect of the present invention is to provide a novel RNase H-dependent isothermal amplification method, wherein an RNase H enzyme or a reverse transcriptase having an RNase H activity is added to an amplification system, and the primers used at least comprise a pair of primers comprising 1 or more core fragments, wherein two adjacent core fragments are separated by 1 to 30 deoxyribonucleobase fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 consists of 1 to 10 ribonucleotides (rNTPs), the number of which is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid.
Further, the reverse transcriptase having RNase H activity is MMLV, HIV DNA polymerase or the like.
Further, the primers used also include a pair of strand displacement primers, one of which is complementary to at least the F3c region at the 3 'end of the target gene and the other of which is complementary to the B3c region at the 5' end of the target gene.
Further, the RNase H-dependent isothermal amplification method comprises the following steps:
step one, extracting genetic materials of a sample to be detected;
designing a pair of primers, wherein the primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1 to 10 ribonucleotides, and the number of the ribonucleotides is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
Further, the RNase H-dependent isothermal amplification method comprises the following steps:
step one, extracting genetic materials of a sample to be detected;
step two, designing a pair of primers and a pair of strand displacement primers; one of the strand displacement primers is at least complementary to the F3c region at the 3 'end of the target gene, and the other strand displacement primer is complementary to the B3c region at the 5' end of the target gene; the primer at least comprises a pair of primers containing 1 or more core fragments, wherein two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base segments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1 to 10 ribonucleotides, and the number of the ribonucleotides is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH 4) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, strand displacement primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
Further, the above strand displacement DNA polymerase includes, but is not limited to, bst DNA polymerase, 3137DNA polymerase, manta DNA polymerase, bsu DNA polymerase, phi 29DNA polymerase.
Further, the isothermal amplification conditions were 60℃for 1 minute for 40 cycles total for amplifying DNA; or, 55 ℃ for 2 minutes; a total of 40 cycles at 60℃for 1 min were used for simultaneous reverse transcription and amplification of RNA.
Further, the amplification system further comprises an RTase, preferably a fluorescent probe or a fluorescent dye.
Further, the amplification system includes a fluorescent probe having a length of 15 to 40 bases, preferably 18 to 30 bases; preferably, the fluorescent probe comprises 1 to 10, preferably 1 to 4 ribonucleotides, which ribonucleotides are one or more of rATP, rCTP, rGTP and rUTP.
Further, the fluorescent probe uses a fluorescent group selected from one or more of the following: FAM, HEX, VIC, ROX, cy3, cy5, NED, etc., the quenching groups employed are selected from one or more of the following: BHQ1, BHQ2, BHQ3, etc.; number of bases between fluorophore and quencher (n): n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, … …; preferably n=1 to 20.
Further, the amplification system comprises the following components in concentration: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, probes 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM.
Further, the amplification system comprises the following components in concentration: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM and fluorescent dye 1×.
Further, the amplification system comprises the following components in concentration: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, probes 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM.
Further, the amplification system comprises the following components in concentration: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, each primer 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM and fluorescent dye 1X.
The second aspect of the invention provides an application of the RNase H-dependent isothermal amplification method in detection or multiplex amplification detection of a target gene of a sample to be detected.
The third aspect of the present invention is to provide the use of the above RNase H-dependent isothermal amplification method for detecting single base mutations and gene polymorphisms.
Compared with the prior art, the invention has the following technical effects:
the invention utilizes the characteristic of RNaseH specific degradation and RNA hybrid chain combined with DNA to open double-chain gaps and accelerate the isothermal amplification process, thereby greatly improving the reaction rate, the specificity and the sensitivity and having very wide application scenes.
Drawings
FIG. 1 is a schematic diagram of the RDA amplification of a DNA template according to one embodiment of the present invention (both the upstream and downstream primers are perfectly complementary to the template);
FIG. 2 is a schematic diagram of the principle of RDA amplification of a DNA template (where a portion of the upstream and downstream primer sequences are not complementary to the target sequence) in one embodiment of the invention;
FIG. 3 is a schematic diagram of the RDA amplification of RNA templates (with both the upstream and downstream primers being perfectly complementary to the templates) according to one embodiment of the invention;
FIG. 4 is a schematic diagram of the RDA amplification of RNA templates (with some of the primers on the upstream and downstream sides being fully complementary to the templates) according to one embodiment of the invention;
FIG. 5 shows the result of HBV detection using the RDA detection system according to one embodiment of the present invention; wherein NC represents a negative control group, and NO-RNase H represents an RNaseH 2-free group;
FIG. 6 shows the result of amplification of HBV DNA template by RDA and LAMP amplification system according to an embodiment of the present invention; wherein, FIG. A, B shows the results of detecting HBV DNA templates by using RNA base modified LAMP primers and RDA primers, respectively, based on RDA technique, and FIG. C, D shows the results of detecting HBV DNA templates by using RNA base modified LAMP primers and conventional LAMP primers, based on LAMP technique;
FIG. 7 shows the results of amplification of JVRNA templates by RDA and LAMP amplification systems according to an embodiment of the invention; wherein, figures A and B show the results of amplifying JEV RNA templates with different LAMP primer combinations, respectively, and figures C and D show the results of amplifying JEV RNA templates with different RDA primer combinations, respectively;
FIG. 8 shows a graph of the results of multiplex RT-RDA detection of the COVID-19 virus, amplification of the 100copies/mLN gene and the E gene.
Detailed Description
The invention provides a novel RNase H-dependent isothermal amplification (RDA), adding RNase H enzyme or reverse transcriptase with RNase H activity into an amplification system, wherein the adopted primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 consists of 1 to 10 ribonucleotides (rNTPs), the number of which is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid.
In a preferred embodiment of the present invention, the reverse transcriptase having RNase H activity is MMLV, HIV DNA polymerase or the like.
In a preferred embodiment of the invention, the RNase H-dependent isothermal amplification method is used for amplifying DNA, and base segment 1 and base segment 2 are complementary to the target sequence. As shown in fig. 1, the principle of the RDA amplification technique in this case is as follows:
step 1, opening a DNA double strand, and respectively binding an upstream primer and a downstream primer (RDA-F and RDA-R) with a DNA target sequence; extending under the action of polymerase to form new double chain. One strand of double-stranded DNA carries RNA (RNA recognition site) at the 5' end.
Step 2, RNase H enzyme recognizes RNA sequence complementary to DNA and cleaves to form a gap.
And 3, continuing extending and displacing the DNA polymerase binding notch, and stripping the original complementary strand to form two new double-stranded DNA and two single-stranded DNA.
Step 4, for the new double strand, the amplification process is cycled to step 1; once every cycle, two new DNA double chains and two single chains are synthesized; the upstream and downstream primers are then combined with the displaced single strand and extended to form a new double-stranded DNA, where only one strand of the double-stranded DNA has RNA at the 5' -end (RNA recognition site).
Step 5, RNase H recognizes the RNA/DNA complex and hydrolyzes the RNA to form a gap.
Step 6, DNA polymerase recognizes the gap extension to form new double-stranded DNA and replaces single strand to form new double-stranded DNA and single-stranded DNA.
Step 7, the primer is combined with the single-stranded DNA to form new double-stranded DNA, and the 5' -ends of both strands are provided with RNA. The digestion and amplification are accelerated and the process is circulated to step 5. So that two double chains and two single chains are formed in each cycle; and repeating the cycle for a plurality of times to achieve exponential amplification.
In a preferred embodiment of the invention, the RNase H-dependent isothermal amplification method is used for amplifying DNA, base segment 1 and base segment 2 being non-complementary to the target sequence. As shown in FIG. 2, in this case, a pair of strand displacement primers (one of which is complementary to at least the F3c region at the 3 'end of the target gene and the other of which is complementary to the B3c region at the 5' end of the target gene) are additionally added to amplify the DNA, and the principle of the RDA amplification DNA template is as follows:
step 1, opening a DNA double chain, complementing a primer with a single-chain DNA template, and synthesizing DNA along the 5'-3' direction under the action of polymerase; the two strand displacement primers displace a single strand under the action of a DNA polymerase.
Step 2, RDA-F and R are combined with single-stranded DNA to form new double-stranded DNA; the double-stranded DNA carries a complex of DNA and RNA. RNase H recognizes and hydrolyzes RNA bound to DNA, creating a gap.
Step 3, DNA polymerase recognizes the notch, and the extension strand replaces and strips the original complementary strand to form two new double-stranded DNA and two single-stranded DNA.
Step 4, RNase H recognizes RNA/DNA complex site, hydrolyzes RNA to form gap, DNA polymerase recognizes gap, extends strand displacement and peels off original complementary strand to form new double-stranded DNA and single-stranded DNA.
Step 5, RDA-F/R is combined with the new single strand and extended under the action of DNA polymerase to form new double-stranded DNA and single-stranded DNA.
The whole process is repeatedly circulated between the steps 4-5. Step 4-5, forming two double chains and two single chains once in each cycle; and repeating the cycle for a plurality of times to achieve exponential amplification.
In a preferred embodiment of the invention, the RNase H-dependent isothermal amplification method is used for amplifying RNA, base segment 1 and base segment 2 being complementary to the target sequence. As shown in FIG. 3, the principle of RDA amplification of RNA templates in this case is as follows:
step 1, RDA-R is complementary to the RNA template and is used for 5'-3' cDNA synthesis under the action of reverse transcriptase (Rtase). RNase H recognizes and hydrolyzes RNA bound to DNA to form single-stranded cDNA.
Step 2, RDA-F is combined with cDNA to synthesize new DNA double strand under the action of DNA polymerase. The DNA polymerase recognizes the nick, displaces the extended strand and strips the original complementary strand to form a new double-stranded DNA and two single-stranded DNAs.
Step 3, RDA-F/R is combined with single strand to synthesize two new double strand DNA. RNase H recognizes RNA/DNA complex and forms a gap in RNA. The DNA polymerase recognizes the nick and extends to form two double stranded DNA and two single stranded DNA.
Step 4, RDA-F/R is combined with single-stranded DNA to form new double-stranded DNA.
The whole process is repeatedly circulated between the steps 2-4. Step 2-4, forming three double chains and 4 single chains once in each cycle; and repeating the cycle for a plurality of times to achieve exponential amplification.
In a preferred embodiment of the invention, the RNase H-dependent isothermal amplification method is used for amplifying RNA, base segment 1 and base segment 2 being non-complementary to the target sequence. As shown in FIG. 4, the principle of RDA amplification of RNA templates in this case is as follows:
step 1, the RDA-R is complementary with the RNA template, and cDNA is synthesized along with 5'-3' under the action of reverse transcriptase to form RNA and DNA complex.
Step 2, RNase H recognizes and hydrolyzes RNA bound to DNA to form single-stranded cDNA. RDA-F specifically binds to cDNA and forms a new DNA duplex along with 5'-3' synthetic DNA by the action of DNA polymerase.
Step 3, RNase H recognizes the RDA-R RNA/DNA complex site and hydrolyzes RNA to form a gap. DNA polymerase recognizes the nick, extends the strand displacement and strips the original complementary strand, forming a new double-stranded DNA and single-stranded DNA.
Step 4, RNase H recognizes the site of the RDA-F RNA/DNA complex, hydrolyzes RNA and forms a notch; DNA polymerase recognizes the nick, extends the strand displacement and strips the original complementary strand, forming a new double-stranded DNA and single-stranded DNA. RDA-R is combined with a new single strand and extended by DNA polymerase to form a new double-stranded DNA.
Step 5, RNase H recognizes the site of RDA-R RNA/DNA complex and selectively hydrolyzes RNA; forming a notch. DNA polymerase recognizes the nick, extends the strand displacement and strips the original complementary strand, forming a new double-stranded DNA and single-stranded DNA.
The whole process is repeatedly circulated between the steps 4-5. Step 4-5, forming two double chains and two single chains once in each cycle; and repeating the cycle for a plurality of times to achieve exponential amplification.
In a preferred embodiment of the present invention, the RNase H-dependent isothermal amplification method comprises the steps of:
step one, extracting genetic materials of a sample to be detected;
designing a pair of primers, wherein the primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1 to 10 ribonucleotides, and the number of the ribonucleotides is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
In a preferred embodiment of the present invention, the RNase H-dependent isothermal amplification method comprises the steps of:
step one, extracting genetic materials of a sample to be detected;
step two, designing a pair of primers and a pair of strand displacement primers; one of the strand displacement primers is at least complementary to the F3c region at the 3 'end of the target gene, and the other strand displacement primer is complementary to the B3c region at the 5' end of the target gene; the primer at least comprises a pair of primers containing 1 or more core fragments, wherein two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base segments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1 to 10 ribonucleotides, and the number of the ribonucleotides is preferably 1 to 4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, strand displacement primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
In a preferred embodiment of the present invention, the DNA polymerase used in the amplification system is a DNA polymerase with a strand displacement function, including but not limited to Bst DNA polymerase, 3137DNA polymerase, manta DNA polymerase, bsu DNA polymerase, phi 29DNA polymerase.
In a preferred embodiment of the invention, isothermal amplification conditions are 60℃for 1 minute for a total of 40 cycles for amplifying DNA; or, 55 ℃ for 2 minutes; a total of 40 cycles at 60℃for 1 min were used for simultaneous reverse transcription and amplification of RNA.
In a preferred embodiment of the invention, the above-mentioned amplification system further comprises an RTase, preferably further comprises a fluorescent probe or a fluorescent dye.
In a preferred embodiment of the present invention, the amplification system comprises a fluorescent probe having a length of 15 to 40 bases, preferably 18 to 30 bases; preferably, the fluorescent probe comprises 1 to 10, preferably 1 to 4 ribonucleotides, which ribonucleotides are one or more of rATP, rCTP, rGTP and rUTP.
In a preferred embodiment of the present invention, the fluorescent probe uses a fluorescent group selected from one or more of the following: FAM, HEX, VIC, ROX, cy3, cy5, NED, etc., the quenching groups employed are selected from one or more of the following: BHQ1, BHQ2, BHQ3, etc.; number of bases between fluorophore and quencher (n): n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, … …; preferably n=1 to 20.
In a preferred embodiment of the present invention, the amplification system described aboveComprises the following components in concentration: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, probes 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM.
In a preferred embodiment of the invention, the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM and fluorescent dye 1×.
In a preferred embodiment of the invention, the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, probes 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM.
In a preferred embodiment of the invention, the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, each primer 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM and fluorescent dye 1X.
The invention also provides application of the RNase H-dependent isothermal amplification method in detection or multiplex amplification detection of target genes of a sample to be detected.
The invention also provides application of the RNase H-dependent isothermal amplification method in detecting single base mutation and gene polymorphism.
The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
In this example, HBV was detected by the RDA detection system, and the primer used was completely paired with the template. The specific experimental design and results are as follows:
1. experiment preparation:
1) RDA primers and probes:
HBV-RDA-F1：CCAACCTCCAATCACrUCACCAACCTCTTGTC(SEQ ID NO.1)；
HBV-RDA-R1：TGGATAGTCCAGAAGArACCAACAAGAAGATGA(SEQ ID NO.2)；
HBV-RDA-P1:FAM-GATGTGTCTGCGGCGTrUrUTATCATATTCCTCT-BHQ 1(SEQ ID NO.3)。
remarks: rU and rA are RNA base modifications (NTPs).
2) HBV DNA template:
HBV whole genome plasmid was given away by third people hospital clinical laboratory in Nantong. Diluted to 10 after quantitative PCR 2 copies/mL～10 7 The copies/mL is ready for use.
3) The RDA amplification system (25. Mu.L) contained:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, HBV-RDA-F1.0. Mu.M, HBV-RDA-R1.0. Mu.M, HBV-RDA-P0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM.
4) RDA amplification procedure: 60 ℃ for 1 minute, and 40 cycles.
2. Experimental results:
gradient diluted HBV DNA templates were detected using RDA (10 2 copies/mL～10 7 cobies/mL); control group without RNaseH2The method comprises the steps of carrying out a first treatment on the surface of the The negative control was not added with DNA template. As shown in FIG. 5, the RDA group amplified HBV DNA template well and negative control had no amplification curve; no RNaseH2 group, no amplification curve; the RDA group has high amplification efficiency, and the Ct value difference between the high-concentration DNA template and the low-concentration DNA template is small.
From the above results, it was found that the primer and the template were completely complementary to each other and amplified well.
Example 2
The present example detects HBV by means of RDA detection system and compares it with conventional LAMP detection technique. The specific experimental design and results are as follows:
lamp primer system:
1) Primer:
HBV-FIP：TGGAATTAGAGGACAAACGGGTGCTGCTATGCCTCATCTT(SEQ ID NO.4)
HBV-BIP：GCTCAAGGAACCTCTATGTTTCGATGATGGGATGGGAATACA(SEQ ID NO.5)
HBV-F3：GGCGTTTTATCATCTTCC(SEQ ID NO.6)
HBV-B3：AGGTTACTTGCGAAAGCC(SEQ ID NO.7)
HBV-LF：TACCTTGATAGTCCAGAAGAACC(SEQ ID NO.8)
HBV-LB：CTACGGACGGAAACTGCAC(SEQ ID NO.9)
2) HBV DNA template:
HBV whole genome plasmid was given away by third people hospital clinical laboratory in Nantong. Diluted to 10 after quantitative PCR 2 copies/mL～10 7 The copies/mL is ready for use.
3) The LAMP amplification system (25. Mu.L) contained:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, FIP and BIP 1.6. Mu.M each, LF and LB 0.4. Mu.M each, B3 and F3 0.2. Mu.M each, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM, SYBR Green (1X).
4) RDA amplification procedure: 60 ℃ for 1 minute, and 40 cycles.
RDA System:
1) Primer(s)
rHBV-FIP：TGGAATTAGAGGACAAACGGGrUGCTGCTATGCCTCATCTT(SEQ ID NO.10)
rHBV-BIP：GCTCAAGGAACCTCTATGTrUTCGATGATGGGATGGGAATACA(SEQ ID NO.11)
HBV-RDA-F2：CCGTTGTTGACGGAATGGTCTrUTGCTGCTATGCCTCATCTT(SEQ ID NO.12)
HBV-RDA-R2：GCTGTGGTGGAATTCCAAACTrUTCGATGATGGGATGGGAATACA(SEQ ID NO.13)
HBV-F3：GGCGTTTTATCATCTTCC(SEQ ID NO.6)
HBV-B3：AGGTTACTTGCGAAAGCC(SEQ ID NO.7)
Remarks: rU is RNA modified, and the underlined sequence is not complementary to HBV sequence.
2) HBV DNA template:
HBV whole genome plasmid was given away by third people hospital clinical laboratory in Nantong. Diluted to 10 after quantitative PCR 2 copies/mL～10 7 The copies/mL is ready for use.
3) The RDA amplification system (25. Mu.L) contained:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, rHBV-FIP and rHBV-BIP (or HBV-RDA-F2 and R2) each 1.0. Mu.M, B3 and F3 each 0.2. Mu.M, RNase H2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM, SYBR Green (1X).
4) RDA amplification procedure: 60 ℃ for 1 minute, and 40 cycles.
3. Analysis of results:
1) RDA amplification efficiency was far higher than LAMP. The amplification efficiency of the LAMP primers FIP and BIP after RNA base modification to form RDA primers rHBV-FIP and rHBV-BIP is completely consistent with that of the RDA primers, as shown in FIG. 6A and FIG. B.
2) The amplification efficiency of the RNA-modified LAMP primers appeared to be better than that of the unmodified primers, see FIGS. 6C and D.
As shown by the experiment, the LAMP primer can be used for RDA primer after being modified by RNA base, and the amplification efficiency is far higher than that of LAMP.
Example 3
The present example uses the RDA detection system to detect encephalitis B virus (Japanese encephalitis virus, JEV) and compares it with conventional LAMP detection techniques. The specific experimental design and results are as follows:
lamp primer system:
1) Primer:
JEV-5-F3：AGCTGGATGGAATGTGAAGG(SEQ ID NO.14)
JEV-5-B3：CTGCAGCATGTCTTCCGT(SEQ ID NO.15)
JEV-5-FIP：CGCAAGTCCCTGCGATGGAAGCTTGCCTGGCCAAAGCAT(SEQ ID NO.16)
JEV-5-BIP：TCAGCAGTGCCAGTAGATTGGGGGTCATCCACTCTCCTTTCG(SEQ ID NO.17)
JEV-5-LF：GGAGTAGCCACATCTGTGCAT(SEQ ID NO.18)
JEV-5-LB：TGCCCACAGGCAGGACA(SEQ ID NO.19)
2) JEVRNA template:
pig encephalitis B virus live vaccine is selected as an RNA template. Diluted to 10 after quantitative PCR 2 copies/mL～10 7 The copies/mL is ready for use.
3) The RT-LAMP amplification system (25. Mu.L) contained:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, FIP and BIP 1.6. Mu.M each, LF and LB 0.4. Mu.M each, B3 and F3 0.2. Mu.M each, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM, SYBR Green (1X).
4) LAMP amplification procedure: 55℃for 2 min (reverse transcription); 60 ℃ for 1 minute, and 40 cycles.
RDA System:
1) Primer(s)
JEV-5-rFIP：CGCAAGTCCCTGCGATGGrArAGCTTGCCTGGCCAAAGCAT(SEQ ID NO.20)
JEV-5-rBIP：TCAGCAGTGCCAGTAGATTGGrGrGGTCATCCACTCTCCTTTCG(SEQ ID NO.21)
Remarks: rU is RNA modified, and the underlined sequence is not complementary to HBV sequence.
2) JEVRNA template:
pig encephalitis B virus live vaccine is selected as an RNA template. Diluted to 10 after quantitative PCR 2 copies/mL～10 7 The copies/mL is ready for use.
3) The RT-RDA amplification system (25. Mu.L) contained:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, rFIP and rBIP each 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM, SYBR Green (1X).
4) RT-RDA amplification procedure: 55℃for 2 min (reverse transcription); 60 ℃ for 1 minute, and 40 cycles.
3. Analysis of results:
experimental results show that the RT-RDA can complete amplification by only one pair of primers, and the amplification efficiency is very high, as shown in FIG. 7C; if the LF and LB-accelerated primers bound to the loop were added to the RT-RDA, the amplification efficiency was rather reduced, see FIG. 7D.
In RT-LAMP, the B3 and F3 strand displacement primers do not appear to be very effective, see FIGS. 7A and B; the amplification efficiency of the two is not greatly different.
As can be seen from the above results, the amplification efficiency of RT-RDA was far higher than that of RT-LAMP.
Example 4
In this example, using a novel coronavirus as an example, multiple RDA systems were tested, and specific experiments and results were as follows:
1. primer and probe:
remarks: the bolded base is RNA modification.
2. Pseudovirus: the COVID-19N gene and E gene pseudoviruses were purchased from Anhui general Biotechnology Inc.
3. Nucleic acid releasing agent: tris 10mM,KCl 10mM,EDTA 1mM,RNA inhibitor 50nM, tritonX-1000.5%.
4. The multiplex RT-RDA amplification system (25. Mu.L) comprises:
Tris-HCl 25mM，KCl 20mM，(NH 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, E1-FIP 1.0. Mu.M, E1-BIP 1.0. Mu.M, N2-FIP 1.0. Mu.M, E1-P0.3. Mu.M, N1-P0.3. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bstDNA polymerase 500nM,MMLV RTase 200nM, thermosensitive UDG 100nM.
RT-RDA amplification procedure: 55℃for 2 min (reverse transcription); 60 ℃ for 1 minute, and 40 cycles.
6. Analysis of results:
multiple RT-RDA, a duplicate amplification system can be easily detected, as shown in FIG. 8.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (15)
1. A novel RNase H-dependent isothermal amplification method is characterized in that an RNase H enzyme or reverse transcriptase with RNase H activity is added into an amplification system, and the adopted primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1-10 ribonucleotides, and the number of the ribonucleotides is preferably 1-4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid.
2. The method of claim 1, wherein the primers further comprise a pair of strand displacement primers, one of which is complementary to at least the F3c region at the 3 'end of the target gene and the other of which is complementary to the B3c region at the 5' end of the target gene.
3. The RNase H dependent isothermal amplification method according to claim 1, wherein the RNase H dependent isothermal amplification method comprises the steps of:
step one, extracting genetic materials of a sample to be detected;
designing a pair of primers, wherein the primers at least comprise a pair of primers containing 1 or more core fragments, and two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1-10 ribonucleotides, and the number of the ribonucleotides is preferably 1-4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
4. The RNase H dependent isothermal amplification method according to claim 1, wherein the RNase H dependent isothermal amplification method comprises the steps of:
step one, extracting genetic materials of a sample to be detected;
step two, designing a pair of primers and a pair of strand displacement primers; one of the strand displacement primers is at least complementary to the F3c region at the 3 'end of the target gene, and the other strand displacement primer is complementary to the B3c region at the 5' end of the target gene; the primer at least comprises a pair of primers containing 1 or more core fragments, wherein two adjacent core fragments are separated by 1-30 deoxyribonucleic acid base fragments; the core fragment comprises at least a 3 base segment portion; from the 5' end, the base segment 1 consists of 10-30 deoxyribonucleotides, and Tm is more than or equal to 35 ℃; base segment 2 is composed of 1-10 ribonucleotides, and the number of the ribonucleotides is preferably 1-4; base segment 3 is composed of 10-30 deoxyribonucleotides and is paired with target sequence nucleic acid;
step three, preparing an amplification system, which comprises: tris-HCl, KCl, (NH) 4 ) 2 SO 4 、MgSO 4 Triton X-100, genetic material of a sample to be tested, dNTPs, primers, RNase H2, RNA inhibitor, strand displacement DNA polymerase and thermosensitive UDG;
and step four, amplifying at constant temperature.
5. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the strand displacement DNA polymerase is selected from Bst DNA polymerase, 3137DNA polymerase, manta DNA polymerase, bsu DNA polymerase, phi 29DNA polymerase.
6. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein isothermal amplification conditions are 60 ℃ for 1 min for 40 cycles total for amplifying DNA; or, 55 ℃ for 2 minutes; a total of 40 cycles at 60℃for 1 min were used for simultaneous reverse transcription and amplification of RNA.
7. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system further comprises an RTase, preferably further comprises a fluorescent probe or a fluorescent dye.
8. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system comprises a fluorescent probe with a length of 15-40 bases, preferably 18-30 bases; preferably, the fluorescent probe comprises 1 to 10, preferably 1 to 4 ribonucleotides, and the ribonucleotides are one or more of rATP, rCTP, rGTP and rUTP.
9. The RNase H dependent isothermal amplification method according to claim 8, wherein the fluorescent probe employs a fluorescent group selected from one or more of the following: FAM, HEX, VIC, ROX, cy3, cy5, NED, etc., the quenching groups employed are selected from one or more of the following: BHQ1, BHQ2, BHQ3, etc.; number of bases between fluorophore and quencher n: n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, … …; preferably n=1 to 20.
10. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, probes 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM.
11. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, primers 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bst DNA polymerase 500nM, thermosensitive UDG 100nM and fluorescent dye 1×.
12. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, each primer 0.1 to 1.0. Mu.M, probe 0.2 to 0.4. Mu.M, RNaseH2200nM, RNA inhibitor 400nM,bst DNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM.
13. The RNase H dependent isothermal amplification method according to claim 4 or 5, wherein the amplification system comprises the following concentrations of components: tris-HCl25mM, KCl 20mM, (NH) 4 ) 2 SO 4 10mM，MgSO 4 4mM, triton X-1000.1% (W/V), dATP 1.6. Mu.M, dGTP 1.6. Mu.M, dCTP 1.6. Mu.M, dTTP 1.6. Mu.M, dUTP 0.5. Mu.M, each primer 0.1 to 1.0. Mu.M, RNaseH2200nM, RNA inhibitor 400nM, bstDNA polymerase 500nM,MMLVRTase 200nM, thermosensitive UDG 100nM and fluorescent dye 1X.
14. Use of an RNase H dependent isothermal amplification method according to any of claims 1-13 for detecting or multiplex amplification of a gene of interest in a sample to be tested.
15. Use of an RNase H dependent isothermal amplification method according to any of claims 1-13 for detecting single base mutations and gene polymorphisms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311005986.7A CN117025730A (en) | 2023-08-10 | 2023-08-10 | Novel RNase H-dependent isothermal amplification method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311005986.7A CN117025730A (en) | 2023-08-10 | 2023-08-10 | Novel RNase H-dependent isothermal amplification method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117025730A true CN117025730A (en) | 2023-11-10 |
Family
ID=88638586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311005986.7A Pending CN117025730A (en) | 2023-08-10 | 2023-08-10 | Novel RNase H-dependent isothermal amplification method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117025730A (en) |
-
2023
- 2023-08-10 CN CN202311005986.7A patent/CN117025730A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3936798B2 (en) | Method for amplifying RNA target sequence | |
| KR101032750B1 (en) | Processes Using Dual Specificity Oligonucleotide and Dual Specificity Oligonucleotide | |
| JP3715657B2 (en) | Methods for subtractive hybridization and difference analysis | |
| EP1423532B1 (en) | Nucleic acid amplification method | |
| US20070059700A1 (en) | Methods and compositions for optimizing multiplex pcr primers | |
| CN114250274A (en) | Amplification of primers with limited nucleotide composition | |
| JP2005523700A (en) | Amplification of DNA to produce a single-stranded product of a given sequence and length | |
| JP2001523471A (en) | Unbiased mRNA amplification method | |
| US6100024A (en) | Methods and compositions for nucleic acid detection by target extension and probe amplification | |
| WO1997004131A1 (en) | Single primer amplification of polynucleotide hairpins | |
| WO2021013257A1 (en) | Crispr multi-target detection method and test kit therefor | |
| WO2023246032A1 (en) | One-pot single-stranded dna cyclization and amplification and crispr/cas-mediated nucleic acid molecule detection method | |
| CN107083427B (en) | DNA ligase mediated DNA amplification technology | |
| EP1682676B1 (en) | Method for amplification of rna sequences | |
| CN116426619B (en) | Multiple target nucleotide detection kit, method and application | |
| CN117025730A (en) | Novel RNase H-dependent isothermal amplification method and application thereof | |
| KR101785687B1 (en) | Method for detection of target nucleic acid sequence by multiple amplification nested signal amplification | |
| CN114369649A (en) | Specific selective amplification and multiplex PCR method and application | |
| JP2024515305A (en) | Nucleic Acid Concentration and Detection | |
| CN113046421B (en) | Method for asymmetrically amplifying target nucleic acid | |
| JP2003510011A (en) | Coupled polymerase chain reaction-restriction endonuclease digestion-ligase detection reaction | |
| JPH06327500A (en) | Method for multiplying nucleic acid sequence and method for detecting same | |
| CN112301101B (en) | CRISPR multi-target detection method and kit thereof | |
| CN117965687A (en) | Amplification method of single-stranded DNA product and application thereof | |
| CN117286223A (en) | One-pot rolling circle transcription and CRISPR/Cas mediated nucleic acid detection method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |