CN106834483A - Detect the template of the allelic ladder of the method and abo blood group locus of abo blood group genotype - Google Patents
Detect the template of the allelic ladder of the method and abo blood group locus of abo blood group genotype Download PDFInfo
- Publication number
- CN106834483A CN106834483A CN201710107151.0A CN201710107151A CN106834483A CN 106834483 A CN106834483 A CN 106834483A CN 201710107151 A CN201710107151 A CN 201710107151A CN 106834483 A CN106834483 A CN 106834483A
- Authority
- CN
- China
- Prior art keywords
- dna fragmentation
- sequence
- dna
- blood group
- abo
- 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.)
- Granted
Links
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
- C12Q1/6876—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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (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 discloses the template of the allelic ladder of the method and abo blood group locus of detection abo blood group genotype.Method provided by the present invention, comprises the following steps：Genomic DNA or STb gene with sample to be tested carries out PCR amplifications as template using primer combination, obtains pcr amplification product, it is then determined which plants allele containing, further determines that the abo blood group genotype of sample to be tested.Primer combination is made up of 6 primers, and nucleotide sequence is followed successively by sequence 1 in sequence table to sequence 6.It is demonstrated experimentally that detecting abo blood group genotype using the method that the present invention is provided, accuracy rate is 100%；The recombinant plasmid composition provided using the present invention as the allelic ladder of abo blood group locus template, the allelic ladder of amplifiable acquisition abo blood group locus, and being used in various commercial reagents boxes.The present invention has important application value.
Description
Technical field
The present invention relates to medical jurisprudence technical field, and in particular to the method and abo blood group gene of detection abo blood group genotype
The template of the allelic ladder of seat.
Background technology
Abo blood group is classical human inheritance's mark, in blood transfusion, paternity test, anthropological studies, legal medical material evidence examination etc.
Field occupies critical role.For abo blood group, traditional serologic test method is that antigen or antibody are tested, and
The most commonly used method is inspection antigenic substance in legal medical material evidence examination, but because abo blood group antigen is glycolipid or glycoprotein,
Easily influenceed by factors such as antigen active, the pollutions of specific and microorganism of antibody when therefore checking.Violate especially for property
Examination of mixed stain in crime, traditional serologic test method also has the limitation that cannot be overcome so that cannot separate women
Victim and the blood group substance of male suspect, the blood group of suspect can only be speculated according to the blood group of victim, once run into
The situation of aggrieved human blood type masking, then cannot accurately infer the blood group of male's material in mixed stain.1985, Gill seminar will
Differential lysis and DNA fingerprint diagram technology are applied to medicolegal examination, and sperm DNA and women in mixed stain are successfully extracted respectively
DNA, solves puzzlement legal medical material evidence examination problem for many years, and this also makes DNA technique check sperm blood group in mixed stain to turn into can
Energy.Nineteen ninety, Yamamoto et al. report the nucleotide sequence of abo blood group gene, accurately detect each allele in DNA
The difference of SNP site base different in sequence, these researchs confirm to be carried out using the difference of SNP site on abo blood group gene
The Genotyping of abo blood group will be a kind of very effective method, especially for micro in medicolegal practice or degraded inspection
Material, but existing abo blood group gene SNP parting still has some shortcomings, for example, cannot repeat；Though can repeat, in capillary
Easily there is bifurcated, acromion, asymmetric etc. phenomenon in peak type on electrophoresis tube platform, leads to not accurately read parting.
The content of the invention
The technical problems to be solved by the invention are how to detect abo blood group genotype.
In order to solve the above technical problems, present invention firstly provides primer combination.
Primer combination provided by the present invention, can be by primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-
R1, primer ABO-R2 and primer ABO-R3 are constituted；
The primer ABO-F1 can be following A1) or A2)：
A1) the single strand dna shown in the sequence 1 in sequence table；
A2 sequence 1) had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1
The DNA molecular of congenerous；
The primer ABO-F2 can be following A3) or A4)：
A3) the single strand dna shown in the sequence 2 in sequence table；
A4 sequence 2) had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2
The DNA molecular of congenerous；
The primer ABO-F3 can be following A5) or A6)：
A5) the single strand dna shown in the sequence 3 in sequence table；
A6 sequence 3) had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3
The DNA molecular of congenerous；
The primer ABO-R1 can be following A7) or A8)：
A7) the single strand dna shown in the sequence 4 in sequence table；
A8 sequence 4) had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4
The DNA molecular of congenerous；
The primer ABO-R2 can be following A9) or A10)：
A9) the single strand dna shown in the sequence 5 in sequence table；
A10) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5
The DNA molecular of identical function；
The primer ABO-R3 can be following A11) or A12)：
A11) the single strand dna shown in the sequence 6 in sequence table；
A12) sequence 6 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6
The DNA molecular of identical function.
The application of the primer combination falls within protection scope of the present invention.The application of primer combination can for (b1) or
(b2)：
(b1) kit for detecting abo blood group genotype is prepared；
(b2) abo blood group genotype is detected.
Kit containing primer combination falls within protection scope of the present invention.The kit can be used to detect
Abo blood group genotype.
The preparation method of the kit containing primer combination falls within protection scope of the present invention.Contain the primer
The preparation method of the kit of combination, it may include the step of individually packing each bar primer.
In order to solve the above technical problems, present invention also offers the method for detection abo blood group genotype.
The method of detection abo blood group genotype provided by the present invention, concretely method one, comprises the following steps：With
The genomic DNA or STb gene of sample to be tested are template, and PCR amplifications are carried out using primer combination, obtain pcr amplification product,
Then make the following judgment：
(d1) if in the pcr amplification product contain DNA fragmentation first and DNA fragmentation fourth, and do not contain DNA fragmentation second and
DNA fragmentation third, then the abo blood group genotype of sample to be tested is AA；
(d2) if containing the DNA fragmentation first, the DNA fragmentation second and the DNA fragmentation fourth in pcr amplification product,
And not containing the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AB；
(d3) if containing the DNA fragmentation first, the DNA fragmentation third and the DNA fragmentation fourth in pcr amplification product,
And not containing the DNA fragmentation second, then the abo blood group genotype of sample to be tested is AO；
(d4) if containing the DNA fragmentation second and the DNA fragmentation fourth in pcr amplification product, and the DNA is not contained
Fragment first and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is BB；
(d5) if containing the DNA fragmentation first, the DNA fragmentation second, the and of the DNA fragmentation third in pcr amplification product
The DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is BO；
(d6) if containing the DNA fragmentation first and the DNA fragmentation third in pcr amplification product, and the DNA is not contained
Fragment second and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is OO；
The nucleotide sequence of the DNA fragmentation first is as shown in the sequence 7 in sequence table；
The nucleotide sequence of the DNA fragmentation second is as shown in the sequence 8 in sequence table；
The nucleotide sequence of the DNA fragmentation third is as shown in the sequence 9 in sequence table；
The nucleotide sequence of the DNA fragmentation fourth is as shown in the sequence 10 in sequence table.
In the above method, the reaction system of described " entering performing PCR amplification " can be by KCl, MgCl2, bovine serum albumin(BSA),
Tween-20, glycerine, NaN3, dNTP, primer mixture, Taq gold medals enzyme, template and Tris-HCl buffer solutions composition；Primer is mixed
Compound is the mixture of each bar primer composition in the primer combination.In reaction system, the concentration of KCl is concretely
50mM, MgCl2Concentration concretely 1.6mM, the concentration of bovine serum albumin(BSA) concretely 0.8mg/mL, Tween-20's is dense
Degree concretely 0.2% (v/v), the concentration of glycerine concretely 3.2% (v/v), NaN3Concentration concretely 0.02% (v/
V), the concentration of dATP, dTTP, dGTP and dCTP can be specifically 200 μM, the primer ABO-F1, the primer ABO-F2, institute
Stating the concentration of primer ABO-F3, the primer ABO-R1, the primer ABO-R2 and the primer ABO-R3 can be specifically
0.32 μM, the concentration of Taq gold medal enzymes concretely 0.1U/ μ L, template concretely 0.05ng/ μ L, Tris-HCl buffer solutions
The Tris-HCl of concentration concretely pH8.3,20mM.
In the above method, the response procedures of described " entering performing PCR amplification " are concretely：95 DEG C of predegeneration 11min；94 DEG C of changes
Property 30s, 59 DEG C annealing 2min, 72 DEG C extension 1min, 28 times circulation；60 DEG C of extension 60min；4 DEG C of preservations.
The method of detection abo blood group genotype provided by the present invention, concretely method two, comprise the following steps：Inspection
Survey in the genomic DNA or STb gene of sample to be tested and whether contain the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation
Third or described DNA fragmentation fourth, then makes the following judgment：
(e1) if containing the DNA fragmentation first and the DNA fragmentation fourth in the genomic DNA or STb gene of sample to be tested,
And not containing the DNA fragmentation second and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AA；
(e2) if in the genomic DNA or STb gene of sample to be tested containing the DNA fragmentation first, the DNA fragmentation second and
The DNA fragmentation fourth, and do not contain the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AB；
(e3) if containing the DNA fragmentation first, the and of the DNA fragmentation third in the genomic DNA or STb gene of sample to be tested
The DNA fragmentation fourth, and do not contain the DNA fragmentation second, then the abo blood group genotype of sample to be tested is AO；
(e4) if containing the DNA fragmentation second and the DNA fragmentation fourth in the genomic DNA or STb gene of sample to be tested,
And not containing the DNA fragmentation first and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is BB；
(e5) if in the genomic DNA or STb gene of sample to be tested containing the DNA fragmentation first, the DNA fragmentation second,
The DNA fragmentation third and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is BO；
(e6) if containing the DNA fragmentation first and the DNA fragmentation third in the genomic DNA or STb gene of sample to be tested,
And not containing the DNA fragmentation second and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is OO.
In order to solve the above technical problems, present invention also offers a kind of allelic gene typing as abo blood group locus
The DNA composition of the template of reference material, it contains the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and described
DNA fragmentation fourth.
The DNA composition specifically can be by the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and described
DNA fragmentation fourth is constituted.
In the DNA composition, the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and the DNA pieces
The mass ratio of Duan Ding can be 1：(0.8~1.2)：(0.8~1.2)：(0.8~1.2).
In the DNA composition, the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and the DNA pieces
The mass ratio of Duan Ding concretely 1：1：1：1.
In order to solve the above technical problems, present invention also offers a kind of allelic gene typing as abo blood group locus
The recombinant plasmid composition of the template of reference material.The recombinant plasmid composition is prepared via a method which：By the DNA
Fragment first, the DNA fragmentation second, the DNA fragmentation third and the DNA fragmentation fourth are inserted respectively into 4 carriers, obtain 4
Recombinant vector；4 recombinant vectors are mixed, recombinant plasmid composition is obtained；
In the recombinant plasmid composition, the carrier can be cloning vector.The cloning vector concretely plasmid
pMD18-T。
The application of the DNA composition or the recombinant plasmid composition falls within protection scope of the present invention.The DNA
The application of composition or the recombinant plasmid composition be a1) a2) or a3)：
A1) as abo blood group locus allelic ladder template；
A2 the allelic ladder of abo blood group locus) is prepared；
A3 the allelic gene typing of abo blood group locus) is detected.
The DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and the DNA fragmentation fourth can be with the bases of people
Because group DNA is template, performing PCR amplification is entered using primer combination (each primer is without fluorescent decoration) and is obtained.
5 ' the ends of the primer ABO-F1, the primer ABO-F2 and the primer ABO-F3 can repair by fluorescence
Decorations, in order to carry out capillary electrophoresis detection to pcr amplification product.The primer ABO-F1, the primer ABO-F2 and described
5 ' the ends of primer ABO-F3 specifically can be modified by TAMRA.
It is demonstrated experimentally that the primer combine detection abo blood group genotype provided using the present invention, accuracy rate is 100%；Using
The present invention provide recombinant plasmid composition as the allelic ladder of abo blood group locus template, it is amplifiable
The allelic ladder of abo blood group locus is obtained, and can be used in various commercial reagents boxes.The present invention has weight
The application value wanted.
Brief description of the drawings
Fig. 1 is the experimental result of the step 2 of embodiment 1.
Fig. 2 is the experimental result of the step 3 of embodiment 3.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, unless otherwise specified, is conventional method.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
The product of Material Evidence Identification Center, Ministry of Public Security is designated as in Typer500.Deionized formamide is the product of ABI companies, is produced
Product catalog number (Cat.No.) is 4311320.ABI3130xl genetic analyzers and Nanodrop1000 micro-spectrophotometers are ABI companies
Product.Plasmid pMD18-T is the product of TaKaRa companies, and catalog number is 6011.
The genotype of embodiment 1, the allele of detection abo blood group locus
First, the preparation of primer combination
Primer combination for detecting the genotype of the allele of abo blood group locus is as follows：
Primer ABO-F1：(5 ' ends carry out TAMRA and repair 5 '-TAMRA-ACACCCCGGAAGGATGTCCTCGTGGTGA-3 '
Decorations) (sequence 1 in sequence table)；
Primer ABO-F2：5 '-TAMRA-GGAAGGATGTCCTCGTGGTA-3 ' (5 ' ends carry out TAMRA modifications) (sequence
Sequence 2 in table)；
Primer ABO-F3：5 '-TAMRA-AGTGGACGTGGACATGGAGTTCC-3 ' (5 ' ends carry out TAMRA modifications)
(sequence 3 in sequence table)；
Primer ABO-R1：5 '-AATGTCCACAGTCACTCGCCACT-3 ' (sequence 4 in sequence table)；
Primer ABO-R2：5 '-CCCGAAGAACCCCCCCAG-3 ' (sequence 5 in sequence table)；
Primer ABO-R3：5 '-TTTTTCGAAGAACGCCCCCAT-3 ' (sequence 6 in sequence table).
Artificial synthesized primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer
ABO-R3。
2nd, the genotype of the allele of detection abo blood group locus
Sample to be tested is sample one, sample two, sample three, sample four, sample five or sample six：
Sample one：The blood of the people that blood group genotype is AA is identified；
Sample two：The blood of the people that blood group genotype is AB is identified；
Sample three：The blood of the people that blood group genotype is AO is identified；
Sample four：The blood of the people that blood group genotype is BB is identified；
Sample five：The blood of the people that blood group genotype is BO is identified；
Sample six：The blood of the people that blood group genotype is OO is identified.
1st, as template, the primer combination prepared using step one carries out PCR amplifications to the genomic DNA with sample to be tested, obtains
To pcr amplification product.
Reaction system is by KCl, MgCl2, bovine serum albumin(BSA), Tween-20, glycerine, NaN3, dNTP, primer mixture,
Taq gold medals enzyme, template and Tris-HCl buffer solutions composition；Primer mixture is each bar in the primer combination of step one preparation
The mixture of primer composition.In reaction system, the concentration of KCl is 50mM, MgCl2Concentration be 1.6mM, bovine serum albumin(BSA)
Concentration is 0.8mg/mL, and the concentration of Tween-20 is 0.2% (v/v), and the concentration of glycerine is 3.2% (v/v), NaN3Concentration be
The concentration of 0.02% (v/v), dATP, dTTP, dGTP and dCTP is 200 μM, primer ABO-F1, primer ABO-F2, primer
The concentration of ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 is 0.32 μM, and the concentration of Taq gold medal enzymes is
0.1U/ μ L, template is the Tris-HCl of pH8.3,20mM for the concentration of 0.05ng/ μ L, Tris-HCl buffer solutions.
Response procedures：95 DEG C of predegeneration 11min；94 DEG C of denaturation 30s, 59 DEG C of annealing 2min, 72 DEG C of extension 1min, follow for 28 times
Ring；60 DEG C of extension 60min；4 DEG C of preservations.
2nd, after completing step 1, to being added in deionized formamide in 1 μ L pcr amplification products and 0.2 μ L Typer500
Mark, is then settled to 20 μ L with deionized formamide, obtains reaction solution.
3rd, after completing step 2, extract reaction solution, 95 DEG C of denaturation 5min are transferred quickly to -20 DEG C and place 5min, Ran Houyong
ABI3130xl genetic analyzers carry out capillary electrophoresis detection, obtain DNA detection collection of illustrative plates.Deposition condition is：Sample introduction voltage
1.2kV, sample injection time is 18s.
Experimental result is shown in that (A is sample one to Fig. 1, and B is sample two, and C is sample three, and D is sample four, and E is sample five, and F is sample
This is six).Result shows that abo blood group locus has 4 allele, and (size is 187bp, core to be respectively designated as allele 1
Nucleotide sequence is as shown in sequence 7 in sequence table), (size is 190bp to allele 2, sequence 8 in nucleotide sequence such as sequence table
It is shown), (size is for allele 3 (size is 192bp, and nucleotide sequence is as shown in sequence 9 in sequence table) and allele 4
200bp, nucleotide sequence is as shown in sequence 10 in sequence table).4 allele 6 blood group genotypes of correspondence：Blood group genotype
To contain allele 1 and allele 4 in the sample one of AA；Blood group genotype for AB sample two in containing allele 1,
Allele 2 and allele 4；Blood group genotype for AO sample three in contain allele 1, allele 3 and equipotential base
Because of 4；Blood group genotype for BB sample four in contain allele 2 and allele 4；Blood group genotype is in the sample five of BO
Contain allele 1, allele 2, allele 3 and allele 4；Blood group genotype for OO sample six in contain equipotential
Gene 1 and allele 3.
The method of embodiment 2, detection abo blood group genotype
First, the method for detecting abo blood group genotype
The detectable abo blood group genotype of primer combination prepared using the step one of embodiment 1.Concretely comprise the following steps：With to be measured
The genomic DNA or STb gene of sample are template, and the primer combination prepared using step one carries out PCR amplifications, obtains PCR amplifications
Product, then according to the nucleotide sequence of pcr amplification product, it is determined that containing allele 1, allele 2, the and of allele 3
Which kind in allele 4, then makes the following judgment：If containing allele 1 and allele 4, sample to be tested
Abo blood group genotype be AA；If containing allele 1, allele 2 and allele 4, the ABO blood of sample to be tested
Type genotype is AB；If containing allele 1, allele 3 and allele 4, the abo blood group genotype of sample to be tested
It is AO；If containing allele 2 and allele 4, the abo blood group genotype of sample to be tested is BB；If containing equipotential
Gene 1, allele 2, allele 3 and allele 4, then the abo blood group genotype of sample to be tested is BO；If containing etc.
Position gene 1 and allele 3, then the abo blood group genotype of sample to be tested is OO.
2nd, accuracy
1st, for template, prepared using the step one of embodiment 1 with 90 blood cards (circle, diameter is 1.0mm) to be measured respectively
Primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 enter performing PCR expansion
Increase, obtain pcr amplification product.
Reaction system is by KCl, MgCl2, bovine serum albumin(BSA), Tween-20, glycerine, NaN3, dNTP, primer mixture,
Taq gold medals enzyme, template and Tris-HCl buffer solutions composition；Primer mixture is the mixture of above-mentioned each bar primer composition.Instead
Answer in system, the concentration of KCl is 50mM, MgCl2Concentration be 1.6mM, the concentration of bovine serum albumin(BSA) is 0.8mg/mL,
The concentration of Tween-20 is 0.2% (v/v), and the concentration of glycerine is 3.2% (v/v), NaN3Concentration be 0.02% (v/v),
The concentration of dATP, dTTP, dGTP and dCTP is 200 μM, and the concentration of each bar primer is 0.32 μM, Taq gold medal enzymes it is dense
It is 0.1U/ μ L to spend, and template is 1 blood card to be measured, and the concentration of Tris-HCl buffer solutions is the Tris-HCl of pH8.3,20mM.
Response procedures：95 DEG C of predegeneration 11min；94 DEG C of denaturation 30s, 59 DEG C of annealing 2min, 72 DEG C of extension 1min, follow for 28 times
Ring；60 DEG C of extension 60min；4 DEG C of preservations.
2nd, after completing step 1, to being added in deionized formamide in 1 μ L pcr amplification products and 0.2 μ L Typer500
Mark, is then settled to 20 μ L with deionized formamide, obtains reaction solution.
3rd, after completing step 2, extract reaction solution, 95 DEG C of denaturation 5min are transferred quickly to -20 DEG C and place 5min, Ran Houyong
ABI3130xl genetic analyzers carry out capillary electrophoresis detection, obtain DNA detection collection of illustrative plates；Then the conclusion according to step one is true
The blood group genotype of fixed blood card to be measured.Deposition condition is：Sample introduction voltage 1.2kV, sample injection time is 18s.
90 blood group genotypes of blood card to be measured are detected using classical serological method.
Experimental result is shown in Table 1.Result shows that the method provided using step one detects abo blood group genotype, and accuracy rate is
100%.
Table 1
The preparation and checking of embodiment 3, the template of the allelic ladder of abo blood group locus
1st, the acquisition of recombinant plasmid
(1) artificial-synthetic DNA's fragment first, DNA fragmentation second, DNA fragmentation third and DNA fragmentation fourth.The nucleotides of DNA fragmentation first
Sequence is as shown in the sequence 7 in sequence table.The nucleotide sequence of DNA fragmentation second is as shown in the sequence 8 in sequence table.DNA fragmentation
Third nucleotide sequence is as shown in the sequence 9 in sequence table.The nucleotide sequence of DNA fragmentation fourth such as the institute of sequence 10 in sequence table
Show.
(2) 4 recombinant plasmids shown in table 2 are prepared, 4 allele comprising abo blood group locus in embodiment 1.
According to sequencing result, the details of each recombinant plasmid are as follows：
Recombinant plasmid first：DNA fragmentation first and plasmid the pMD18-T connection that step (1) is synthesized, obtain recombinant plasmid first；
Recombinant plasmid second：DNA fragmentation second and plasmid the pMD18-T connection that step (1) is synthesized, obtain recombinant plasmid second；
Recombinant plasmid third：DNA fragmentation third and plasmid the pMD18-T connection that step (1) is synthesized, obtain recombinant plasmid third；
Recombinant plasmid fourth：DNA fragmentation fourth and plasmid the pMD18-T connection that step (1) is synthesized, obtain recombinant plasmid fourth.
Table 2
| Numbering | Locus | Allele | Recombinant plasmid title |
| 1 | Abo blood group locus | 1 | Recombinant plasmid first |
| 2 | Abo blood group locus | 2 | Recombinant plasmid second |
| 3 | Abo blood group locus | 3 | Recombinant plasmid third |
| 4 | Abo blood group locus | 4 | Recombinant plasmid fourth |
2nd, the preparation of the template of the allelic ladder of abo blood group locus
(1) recombinant plasmid obtained in simultaneously dilution step 1 is measured respectively using Nanodrop1000 micro-spectrophotometers
The concentration of DNA obtains the dilution of each recombinant plasmid to 1ng/ μ L.
(2) after completing step (1), the μ L of dilution 1 for taking each recombinant plasmid are mixed, and are then settled to ultra-pure water
1mL, obtains the template of the allelic ladder of abo blood group locus.
In the template of the allelic ladder of abo blood group locus, the DNA of each allele is dense in step one
Degree is 1pg/ μ L.
3rd, the checking of the template of the allelic ladder of abo blood group locus
(1) using the template of the allelic ladder of abo blood group locus as template, using the step of embodiment 1
The one primer ABO-F1 for preparing, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 enter
Performing PCR is expanded, and obtains pcr amplification product.
Reaction system and response procedures are with the step 2 of embodiment 11.
(2) after completing step (1), to adding 1 μ L pcr amplification products and 0.2 μ L Typer500 in deionized formamide
Internal standard, is then settled to 20 μ L with deionized formamide, obtains reaction solution.
(3) after completing step (2), extract reaction solution, 95 DEG C of denaturation 5min are transferred quickly to -20 DEG C and place 5min, Ran Houyong
ABI3130xl genetic analyzers carry out capillary electrophoresis detection, obtain DNA detection collection of illustrative plates.Deposition condition is：Sample introduction voltage
1.2kV, sample injection time is 18s.
Experimental result is shown in Fig. 2.Result shows that each allelic gene typing of abo blood group locus is complete, correct, peak type point
It is sharp clear, it is harmonious good without miscellaneous peak.Therefore, can be with using the template of the allelic ladder of abo blood group locus
Prepare the allelic ladder of abo blood group locus.
<110>Material Evidence Identification Center, Ministry of Public Security
<120>Detect the template of the allelic ladder of the method and abo blood group locus of abo blood group genotype
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
acaccccgga aggatgtcct cgtggtga 28
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
ggaaggatgt cctcgtggta 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
agtggacgtg gacatggagt tcc 23
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
aatgtccaca gtcactcgcc act 23
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
cccgaagaac ccccccag 18
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
tttttcgaag aacgccccca t 21
<210> 7
<211> 187
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
agtggacgtg gacatggagt tccgcgacca cgtgggcgtg gagatcctga ctccgctgtt 60
cggcaccctg caccccggct tctacggaag cagccgggag gccttcacct acgagcgccg 120
gccccagtcc caggcctaca tccccaagga cgagggcgat ttctactacc tgggggggtt 180
cttcggg 187
<210> 8
<211> 190
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
agtggacgtg gacatggagt tccgcgacca cgtgggcgtg gagatcctga ctccgctgtt 60
cggcaccctg caccccggct tctacggaag cagccgggag gccttcacct acgagcgccg 120
gccccagtcc caggcctaca tccccaagga cgagggcgat ttctactaca tgggggcgtt 180
cttcgaaaaa 190
<210> 9
<211> 192
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
ggaaggatgt cctcgtggta ccccttggct ggctcccatt gtctgggagg gcacattcaa 60
catcgacatc ctcaacgagc agttcaggct ccagaacacc accattgggt taactgtgtt 120
tgccatcaag aagtaagtca gtgaggtggc cgagggtaga gacccaggca gtggcgagtg 180
actgtggaca tt 192
<210> 10
<211> 200
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
acaccccgga aggatgtcct cgtggtgacc ccttggctgg ctcccattgt ctgggagggc 60
acattcaaca tcgacatcct caacgagcag ttcaggctcc agaacaccac cattgggtta 120
actgtgtttg ccatcaagaa gtaagtcagt gaggtggccg agggtagaga cccaggcagt 180
ggcgagtgac tgtggacatt 200
Claims (10)
1. primer combination, by primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer
ABO-R3 is constituted；
The primer ABO-F1 is following A1) or A2)：
A1) the single strand dna shown in the sequence 1 in sequence table；
A2 sequence 1) had into identical work(by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1
The DNA molecular of energy；
The primer ABO-F2 is following A3) or A4)：
A3) the single strand dna shown in the sequence 2 in sequence table；
A4 sequence 2) had into identical work(by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2
The DNA molecular of energy；
The primer ABO-F3 is following A5) or A6)：
A5) the single strand dna shown in the sequence 3 in sequence table；
A6 sequence 3) had into identical work(by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3
The DNA molecular of energy；
The primer ABO-R1 is following A7) or A8)：
A7) the single strand dna shown in the sequence 4 in sequence table；
A8 sequence 4) had into identical work(by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4
The DNA molecular of energy；
The primer ABO-R2 is following A9) or A10)：
A9) the single strand dna shown in the sequence 5 in sequence table；
A10) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 identical
The DNA molecular of function；
The primer ABO-R3 is following A11) or A12)：
A11) the single strand dna shown in the sequence 6 in sequence table；
A12) sequence 6 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6 identical
The DNA molecular of function.
2. the application of primer combination described in claim 1, is (b1) or (b2)：
(b1) kit for detecting abo blood group genotype is prepared；
(b2) abo blood group genotype is detected.
3. the kit of primer combination described in claim 1 is contained；The kit is used to detect abo blood group genotype.
4. the preparation method of kit described in claim 3, including the step of each bar primer is individually packed.
5. a kind of method for detecting abo blood group genotype, comprises the following steps：Genomic DNA or STb gene with sample to be tested are
Template, PCR amplifications are carried out using primer combination described in claim 1, obtain pcr amplification product, are then made the following judgment：
(d1) if containing DNA fragmentation first and DNA fragmentation fourth in the pcr amplification product, and DNA fragmentation second and DNA are not contained
Fragment third, then the abo blood group genotype of sample to be tested is AA；
(d2) if containing the DNA fragmentation first, the DNA fragmentation second and the DNA fragmentation fourth in pcr amplification product, and not
Containing the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AB；
(d3) if containing the DNA fragmentation first, the DNA fragmentation third and the DNA fragmentation fourth in pcr amplification product, and not
Containing the DNA fragmentation second, then the abo blood group genotype of sample to be tested is AO；
(d4) if containing the DNA fragmentation second and the DNA fragmentation fourth in pcr amplification product, and the DNA fragmentation is not contained
First and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is BB；
(d5) if containing the DNA fragmentation first, the DNA fragmentation second, the DNA fragmentation third and described in pcr amplification product
DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is BO；
(d6) if containing the DNA fragmentation first and the DNA fragmentation third in pcr amplification product, and the DNA fragmentation is not contained
Second and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is OO；
The nucleotide sequence of the DNA fragmentation first is as shown in the sequence 7 in sequence table；
The nucleotide sequence of the DNA fragmentation second is as shown in the sequence 8 in sequence table；
The nucleotide sequence of the DNA fragmentation third is as shown in the sequence 9 in sequence table；
The nucleotide sequence of the DNA fragmentation fourth is as shown in the sequence 10 in sequence table.
6. a kind of method for detecting abo blood group genotype, comprises the following steps：Detect the genomic DNA or STb gene of sample to be tested
In whether contain DNA fragmentation first, DNA fragmentation second, DNA fragmentation third or DNA fragmentation fourth, then make the following judgment：
(e1) if containing the DNA fragmentation first and the DNA fragmentation fourth in the genomic DNA or STb gene of sample to be tested, and not
Containing the DNA fragmentation second and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AA；
(e2) if containing the DNA fragmentation first, the DNA fragmentation second and described in the genomic DNA or STb gene of sample to be tested
DNA fragmentation fourth, and do not contain the DNA fragmentation third, then the abo blood group genotype of sample to be tested is AB；
(e3) if containing the DNA fragmentation first, the DNA fragmentation third and described in the genomic DNA or STb gene of sample to be tested
DNA fragmentation fourth, and do not contain the DNA fragmentation second, then the abo blood group genotype of sample to be tested is AO；
(e4) if containing the DNA fragmentation second and the DNA fragmentation fourth in the genomic DNA or STb gene of sample to be tested, and not
Containing the DNA fragmentation first and the DNA fragmentation third, then the abo blood group genotype of sample to be tested is BB；
(e5) if containing the DNA fragmentation first, the DNA fragmentation second, described in the genomic DNA or STb gene of sample to be tested
DNA fragmentation third and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is BO；
(e6) if containing the DNA fragmentation first and the DNA fragmentation third in the genomic DNA or STb gene of sample to be tested, and not
Containing the DNA fragmentation second and the DNA fragmentation fourth, then the abo blood group genotype of sample to be tested is OO；
The nucleotide sequence of the DNA fragmentation first is as shown in the sequence 7 in sequence table；
The nucleotide sequence of the DNA fragmentation second is as shown in the sequence 8 in sequence table；
The nucleotide sequence of the DNA fragmentation third is as shown in the sequence 9 in sequence table；
The nucleotide sequence of the DNA fragmentation fourth is as shown in the sequence 10 in sequence table.
7. a kind of DNA composition of the template of allelic ladder as abo blood group locus, contains DNA fragmentation
First, DNA fragmentation second, DNA fragmentation third and DNA fragmentation fourth；
The nucleotide sequence of the DNA fragmentation first is as shown in the sequence 7 in sequence table；
The nucleotide sequence of the DNA fragmentation second is as shown in the sequence 8 in sequence table；
The nucleotide sequence of the DNA fragmentation third is as shown in the sequence 9 in sequence table；
The nucleotide sequence of the DNA fragmentation fourth is as shown in the sequence 10 in sequence table.
8. a kind of recombinant plasmid composition of the template of allelic ladder as abo blood group locus, its feature
It is：The recombinant plasmid composition is prepared via a method which：By DNA fragmentation first, DNA fragmentation second, the and of DNA fragmentation third
DNA fragmentation fourth is inserted respectively into 4 carriers, obtains 4 recombinant vectors；4 recombinant vectors are mixed, obtains recombinating matter
Grain composition；
The nucleotide sequence of the DNA fragmentation first is as shown in the sequence 7 in sequence table；
The nucleotide sequence of the DNA fragmentation second is as shown in the sequence 8 in sequence table；
The nucleotide sequence of the DNA fragmentation third is as shown in the sequence 9 in sequence table；
The nucleotide sequence of the DNA fragmentation fourth is as shown in the sequence 10 in sequence table.
9. recombinant plasmid composition as claimed in claim 8, it is characterised in that：The carrier is cloning vector.
10. DNA composition described in claim 7 or, the application of recombinant plasmid composition described in claim 8 or 9, be a1) or
A2) or a3)：
A1) as abo blood group locus allelic ladder template；
A2 the allelic ladder of abo blood group locus) is prepared；
A3 the allelic gene typing of abo blood group locus) is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710107151.0A CN106834483B (en) | 2017-02-27 | 2017-02-27 | Method for detecting ABO blood group genotype and template of allelic typing standard of ABO blood group locus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710107151.0A CN106834483B (en) | 2017-02-27 | 2017-02-27 | Method for detecting ABO blood group genotype and template of allelic typing standard of ABO blood group locus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106834483A true CN106834483A (en) | 2017-06-13 |
| CN106834483B CN106834483B (en) | 2020-05-12 |
Family
ID=59133684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710107151.0A Active CN106834483B (en) | 2017-02-27 | 2017-02-27 | Method for detecting ABO blood group genotype and template of allelic typing standard of ABO blood group locus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106834483B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042165A (en) * | 2019-03-21 | 2019-07-23 | 苏州西山生物技术有限公司 | Macaque abo blood group methods of genotyping |
| CN110942806A (en) * | 2018-09-25 | 2020-03-31 | 深圳华大法医科技有限公司 | Blood type genotyping method and device and storage medium |
| CN112680547A (en) * | 2020-03-13 | 2021-04-20 | 苏州白垩纪生物科技有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit and application thereof |
| CN115725711A (en) * | 2022-08-16 | 2023-03-03 | 深圳市血液中心(深圳市输血医学研究所) | Amplification primer group of blood group antigen coding gene in frozen whole blood, amplification method and genotyping method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900314A (en) * | 2006-07-19 | 2007-01-24 | 辽宁省刑事科学技术研究所 | Fluorescence labelling ABO gene typing method and its reagent kit |
| CN101921834A (en) * | 2010-05-17 | 2010-12-22 | 浙江省血液中心 | Polymerase chain reaction-sequence based typing (PCR-SBT) method for ABO blood type genotyping and reagent |
-
2017
- 2017-02-27 CN CN201710107151.0A patent/CN106834483B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900314A (en) * | 2006-07-19 | 2007-01-24 | 辽宁省刑事科学技术研究所 | Fluorescence labelling ABO gene typing method and its reagent kit |
| CN101921834A (en) * | 2010-05-17 | 2010-12-22 | 浙江省血液中心 | Polymerase chain reaction-sequence based typing (PCR-SBT) method for ABO blood type genotyping and reagent |
Non-Patent Citations (6)
| Title |
|---|
| GOTARO WATANABE等: "Amplified product length polymorphism (APLP):a novel strategy for genotyping the ABO blood group", 《HUM GENET》 * |
| HWAN YOUNG LEE等: "Rapid Direct PCR for ABO Blood Typing", 《JOURNAL OF FORENSIC SCIENCES》 * |
| TOMONORI MURO等: "Determination of ABO genotypes by real-time PCR using allele-specific primers", 《LEGAL MEDICINE》 * |
| 何吉等: "基因扫描技术在ABO血型基因型鉴定中的应用", 《中华检验医学杂志》 * |
| 秦斐等: "单管PCR扩增鉴定ABO血型基因型", 《中国实验血液学杂志》 * |
| 陈昆峰等: "《法医检验技术》", 30 September 2007, 海洋出版社 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110942806A (en) * | 2018-09-25 | 2020-03-31 | 深圳华大法医科技有限公司 | Blood type genotyping method and device and storage medium |
| CN110042165A (en) * | 2019-03-21 | 2019-07-23 | 苏州西山生物技术有限公司 | Macaque abo blood group methods of genotyping |
| CN110042165B (en) * | 2019-03-21 | 2022-08-12 | 苏州西山生物技术有限公司 | Macaque ABO blood type genotyping method |
| CN112680547A (en) * | 2020-03-13 | 2021-04-20 | 苏州白垩纪生物科技有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit and application thereof |
| CN112680547B (en) * | 2020-03-13 | 2023-07-07 | 苏州白垩纪生物科技有限公司 | Real-time fluorescent nucleic acid isothermal amplification detection kit and application thereof |
| CN115725711A (en) * | 2022-08-16 | 2023-03-03 | 深圳市血液中心(深圳市输血医学研究所) | Amplification primer group of blood group antigen coding gene in frozen whole blood, amplification method and genotyping method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106834483B (en) | 2020-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106834483A (en) | Detect the template of the allelic ladder of the method and abo blood group locus of abo blood group genotype | |
| Hennequin et al. | Microsatellite typing as a new tool for identification of Saccharomyces cerevisiae strains | |
| Gibson et al. | The human serum resistance associated gene is ubiquitous and conserved in Trypanosoma brucei rhodesiense throughout East Africa | |
| Tavanti et al. | Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III | |
| Graser et al. | Molecular taxonomy of the Trichophyton rubrum complex | |
| Landlinger et al. | Species-specific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology | |
| Echevarrı́a et al. | Screening of active lyssavirus infection in wild bat populations by viral RNA detection on oropharyngeal swabs | |
| CN105567789B (en) | The PCR primer pair composition and its application of identification or auxiliary identification sturgeon germplasm | |
| Calcagno et al. | Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis | |
| WO2012169099A1 (en) | Method for detecting fungi, reaction solution for pcr, and carrier for detecting fungi | |
| Carlotti et al. | Rapid identification and fingerprinting of Candida krusei by PCR-based amplification of the species-specific repetitive polymorphic sequence CKRS-1 | |
| CN108977571A (en) | Identify the fluorescence PCR detection reagent kit and application of four kinds of Araeceae medicinal plants | |
| Csányi et al. | Y‐chromosome analysis of ancient Hungarian and two modern Hungarian‐speaking populations from the Carpathian basin | |
| CN105713990B (en) | Wheat molecular marker and its application in identification wheat yield correlated traits | |
| Romeo et al. | A multiplex PCR protocol for rapid identification of Candida glabrata and its phylogenetically related species Candida nivariensis and Candida bracarensis | |
| CN105177148B (en) | The double PCR primer of two kinds of grape ulcer bacterium of detection and its application simultaneously | |
| Black et al. | Molecular methods to distinguish between classical rabies and the rabies-related European bat lyssaviruses | |
| US7947446B2 (en) | High throughput mutation screening methods and kits using a universalized approach—differential sequence fill-in (DSF)-enabled sequential adapter ligation and amplification | |
| CN105256075B (en) | The fluorescent PCR kit and detection method of eight kinds of Genotypings of hepatitis type B virus | |
| CN101329250B (en) | Method for detecting sequence of PCR-LCR gene polymorphism of fluorescence labeling probe | |
| Martinez‐Culebras et al. | RAPD analysis of Colletotrichum species isolated from strawberry and the design of specific primers for the identification of C. fragariae | |
| CN102409101A (en) | PCR-LDR(Polymerase Chain Reaction and Ligase Detection Reaction) gene polymorphism sequencing method for universal fluorescence labeling probe | |
| CA2371128A1 (en) | Transposon-based genetic marker | |
| CN101633963B (en) | Specific primer for hepatitis B virus YMDD motif mutation detection and liquid phase chip and method thereof | |
| US20220267829A1 (en) | Combinatorial Microarray Assay for Detecting and Genotyping SARS-CoV-2 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |