CN106834483A - Detect the template of the allelic ladder of the method and abo blood group locus of abo blood group genotype - Google Patents

Abstract

The invention discloses a method for detecting the ABO blood group genotype and a template of the allele typing standard of the ABO blood group gene locus. The method provided by the present invention includes the following steps: using the genomic DNA or total DNA of the sample to be tested as a template, using a combination of primers to perform PCR amplification to obtain a PCR amplification product, and then determining which alleles are contained, and further determining The ABO blood type genotype of the sample to be tested. The primer combination is composed of 6 primers, and the nucleotide sequences are sequentially sequence 1 to sequence 6 in the sequence list. Experiments have proved that the accuracy rate is 100% by adopting the method provided by the invention to detect ABO blood type genotype; using the recombinant plasmid composition provided by the invention as the template of the allelic typing standard of ABO blood type locus can be amplified to obtain Allelic ladder for ABO blood group loci and available in a variety of commercial kits. The invention has important application value.

Description

Method for detecting ABO blood group genotype and allelic typing of ABO blood group loci template for standards

technical field

The invention relates to the technical field of forensic medicine, in particular to a method for detecting ABO blood type genotype and a template for an allele typing standard of an ABO blood type locus.

Background technique

ABO blood type is a classic human genetic marker, which plays an important role in blood transfusion, paternity testing, anthropological research, forensic evidence examination and other fields. For ABO blood type, the traditional serological test method is to test antigen or antibody, and the most commonly used method in forensic evidence test is to test antigenic substances, but because the ABO blood group antigen is glycolipid or glycoprotein, it is vulnerable to testing. The influence of factors such as antigen activity, antibody specificity, and microbial contamination. Especially for the mixed spot test in targeted crimes, the traditional serological test method also has insurmountable limitations, making it impossible to separate the blood type substances of the female victim and the male suspect. Blood type, once the victim's blood type is masked, it is impossible to accurately infer the blood type of the male substance in the mixed spot. In 1985, Gill's research group applied the differential lysis method and DNA fingerprinting technology to forensic examination, and successfully extracted sperm DNA and female DNA in mixed plaques, which solved the problem that has plagued forensic evidence examination for many years, which also made DNA technology examination mixed. Sperm blood typing in spots becomes possible. In 1990, Yamamoto et al. reported the nucleotide sequence of the ABO blood type gene, and accurately detected the base differences of the different SNP sites of each allele in the DNA sequence. Differential genotyping of ABO blood type will be a very effective method, especially for trace or degraded samples in forensic practice, but the existing ABO blood type gene SNP typing still has some deficiencies, for example, it cannot be repeated; although It can be repeated, but the peak type on the capillary electrophoresis platform is prone to bifurcation, shoulder, asymmetry, etc., which makes it impossible to accurately read the typing.

Contents of the invention

The technical problem to be solved by the present invention is how to detect the ABO blood type genotype.

In order to solve the above technical problems, the present invention firstly provides a primer combination.

The primer combination provided by the present invention can be composed of primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3;

The primer ABO-F1 may be A1) or A2) as follows:

A1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing;

A2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1;

The primer ABO-F2 can be as follows A3) or A4):

A3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing;

A4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2;

The primer ABO-F3 can be as follows A5) or A6):

A5) a single-stranded DNA molecule shown in sequence 3 in the sequence listing;

A6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 3 and has the same function as sequence 3;

The primer ABO-R1 can be as follows A7) or A8):

A7) a single-stranded DNA molecule shown in sequence 4 in the sequence listing;

A8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 4 and has the same function as sequence 4;

The primer ABO-R2 can be as follows A9) or A10):

A9) the single-stranded DNA molecule shown in sequence 5 in the sequence listing;

A10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 5 and has the same function as sequence 5;

The primer ABO-R3 can be as follows A11) or A12):

A11) the single-stranded DNA molecule shown in sequence 6 in the sequence listing;

A12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 6 and has the same function as sequence 6.

The application of the primer combination also belongs to the protection scope of the present invention. The application of the primer combination can be (b1) or (b2):

(b1) preparing a test kit for detecting ABO blood group genotype;

(b2) Detection of ABO blood group genotype.

The kit containing the primer combination also belongs to the protection scope of the present invention. The kit can be used to detect ABO blood group genotype.

The preparation method of the kit containing the primer combination also belongs to the protection scope of the present invention. The preparation method of the kit containing the primer combination may include the step of packaging each primer individually.

In order to solve the above technical problems, the present invention also provides a method for detecting ABO blood group genotype.

The method for detecting the genotype of ABO blood type provided by the present invention can specifically be method one, comprising the following steps: using the genomic DNA or total DNA of the sample to be tested as a template, and using the primer combination to perform PCR amplification to obtain PCR amplification The product is then judged as follows:

(d1) If the PCR amplification product contains DNA fragment A and DNA fragment D, and does not contain DNA fragment B and DNA fragment C, the ABO blood type genotype of the sample to be tested is AA;

(d2) If the PCR amplification product contains the DNA fragment A, the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment C, the ABO blood type genotype of the sample to be tested is AB;

(d3) If the PCR amplification product contains the DNA fragment A, the DNA fragment C and the DNA fragment D, and does not contain the DNA fragment B, the ABO blood type genotype of the sample to be tested is AO;

(d4) If the PCR amplification product contains the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment A and the DNA fragment C, the ABO blood type genotype of the sample to be tested is BB;

(d5) If the PCR amplification product contains the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D, the ABO blood type genotype of the sample to be tested is BO;

(d6) If the PCR amplification product contains the DNA fragment A and the DNA fragment C, and does not contain the DNA fragment B and the DNA fragment D, the ABO blood type genotype of the sample to be tested is OO;

The nucleotide sequence of the DNA fragment A is shown in sequence 7 in the sequence listing;

The nucleotide sequence of the DNA fragment B is shown in sequence 8 in the sequence listing;

The nucleotide sequence of the DNA fragment C is shown in sequence 9 in the sequence listing;

The nucleotide sequence of the DNA fragment D is shown as sequence 10 in the sequence listing.

In the above method, the reaction system of "performing PCR amplification" can be buffered by KCl, MgCl ₂ , bovine serum albumin, Tween-20, glycerol, NaN ₃ , dNTP, primer mixture, Taq gold brand enzyme, template and Tris-HCl The composition of the liquid; the primer mixture is the mixture composed of each primer in the primer combination. In the reaction system, the concentration of KCl can be specifically 50mM, the concentration of MgCl can be specifically _1.6mM , the concentration of bovine serum albumin can be specifically 0.8mg/mL, and the concentration of Tween-20 can be specifically 0.2% (v/v ), the concentration of glycerol can be 3.2% (v/v), the concentration of NaN ₃ can be 0.02% (v/v), the concentration of dATP, dTTP, dGTP and dCTP can be 200 μ M, and the primer ABO -The concentration of F1, the primer ABO-F2, the primer ABO-F3, the primer ABO-R1, the primer ABO-R2 and the primer ABO-R3 can be 0.32 μM, and the Taq gold enzyme Specifically, the concentration can be 0.1 U/μL, the template can be specifically 0.05 ng/μL, and the concentration of the Tris-HCl buffer can be specifically pH 8.3, 20 mM Tris-HCl.

In the above method, the specific reaction program of "performing PCR amplification" can be: pre-denaturation at 95°C for 11 minutes; denaturation at 94°C for 30 seconds, annealing at 59°C for 2 minutes, extension at 72°C for 1 minute, 28 cycles; extension at 60°C for 60 minutes; 4 Store at ℃.

The method for detecting the genotype of ABO blood group provided by the present invention can specifically be method two, comprising the following steps: detecting whether the genomic DNA or the total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment B, the DNA fragment C or described DNA fragment D, then carry out following judgment:

(e1) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A and the DNA fragment D, and does not contain the DNA fragment B and the DNA fragment C, then the ABO blood group gene of the sample to be tested Type is AA;

(e2) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment C, the ABO blood group gene of the sample to be tested Type is AB;

(e3) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment C and the DNA fragment D, and does not contain the DNA fragment B, then the ABO blood group gene of the sample to be tested Type is AO;

(e4) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment A and the DNA fragment C, then the ABO blood group gene of the sample to be tested Type is BB;

(e5) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D, the ABO blood type genotype of the sample to be tested is BO ;

(e6) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A and the DNA fragment C, and does not contain the DNA fragment B and the DNA fragment D, then the ABO blood group gene of the sample to be tested The type is OO.

In order to solve the above-mentioned technical problems, the present invention also provides a DNA composition as a template for the allelic typing standard of the ABO blood group locus, which contains the DNA fragment A, the DNA fragment B, the DNA Fragment C and the DNA fragment D.

The DNA composition may specifically consist of the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D.

In the DNA composition, the mass ratio of the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D can be 1: (0.8-1.2): (0.8-1.2): ( 0.8～1.2).

In the DNA composition, the mass ratio of the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D may be specifically 1:1:1:1.

In order to solve the above technical problems, the present invention also provides a recombinant plasmid composition as a template for the allelic typing standard of the ABO blood group loci. The recombinant plasmid composition is prepared by the following method: the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D are respectively inserted into 4 vectors to obtain 4 recombinant vectors ; Mixing the 4 recombinant vectors to obtain a recombinant plasmid composition;

In the recombinant plasmid composition, the vector may be a cloning vector. The cloning vector can specifically be the plasmid pMD18-T.

The application of the DNA composition or the recombinant plasmid composition also belongs to the protection scope of the present invention. The application of the DNA composition or the recombinant plasmid composition is a1) or a2) or a3):

a1) as a template for the allelic typing standard of the ABO blood group locus;

a2) preparing the allelic typing standards for ABO blood group loci;

a3) Detecting the allelic typing of the ABO blood group loci.

The DNA Fragment A, the DNA Fragment B, the DNA Fragment C and the DNA Fragment D can all use human genomic DNA as a template, and use the primer combination (each primer is not fluorescently modified) for PCR amplification get.

The 5' ends of the primer ABO-F1, the primer ABO-F2 and the primer ABO-F3 can all be fluorescently modified, so as to facilitate capillary electrophoresis detection of PCR amplification products. Specifically, the 5' ends of the primer ABO-F1, the primer ABO-F2 and the primer ABO-F3 can be modified by TAMRA.

Experiments have proved that the accuracy rate is 100% when the primer combination provided by the invention is used to detect the ABO blood type genotype; the recombinant plasmid composition provided by the invention is used as the template of the allele typing standard of the ABO blood type locus, which can be amplified Allelic ladders for ABO blood group loci are available and available in a variety of commercial kits. The invention has important application value.

Description of drawings

Fig. 1 is the experimental result of step 2 of embodiment 1.

Fig. 2 is the experimental result of step 3 of embodiment 3.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The internal standard of Typer500 is the product of the Physical Evidence Appraisal Center of the Ministry of Public Security. Deionized formamide is a product of ABI Company, the product catalog number is 4311320. Both ABI3130xl Genetic Analyzer and Nanodrop1000 Micro Spectrophotometer are products of ABI Company. Plasmid pMD18-T is a product of TaKaRa Company, the product catalog number is 6011.

Embodiment 1, detect the genotype of the allele of ABO blood type locus

1. Preparation of primer combinations

The primer combinations for detecting the genotypes of the alleles of the ABO blood group locus are as follows:

Primer ABO-F1: 5'-TAMRA-ACACCCCGGAAGGATGTCCTCGTGGTGA-3' (TAMRA modification at the 5' end) (sequence 1 in the sequence listing);

Primer ABO-F2: 5'-TAMRA-GGAAGGATGTCCTCGTGGTA-3' (TAMRA modification at the 5' end) (sequence 2 in the sequence listing);

Primer ABO-F3: 5'-TAMRA-AGTGGACGTGGACATGGAGTTCC-3' (TAMRA modification at the 5' end) (sequence 3 in the sequence listing);

Primer ABO-R1: 5'-AATGTCCACAGTCACTCGCCACT-3' (sequence 4 in the sequence listing);

Primer ABO-R2: 5'-CCCGAAGAACCCCCCCAG-3' (sequence 5 in the sequence listing);

Primer ABO-R3: 5'-TTTTTCGAAGAACGCCCCCAT-3' (sequence 6 in the sequence listing).

The primers ABO-F1, ABO-F2, ABO-F3, ABO-R1, ABO-R2 and ABO-R3 were artificially synthesized.

2. Detect the genotype of the allele of the ABO blood group locus

The sample to be tested is sample 1, sample 2, sample 3, sample 4, sample 5 or sample 6:

Sample 1: the blood of a person whose blood genotype has been identified as AA;

Sample 2: the blood of a person whose blood genotype has been identified as AB;

Sample 3: the blood of a person whose blood genotype has been identified as AO;

Sample 4: the blood of a person whose blood genotype has been identified as BB;

Sample 5: the blood of a person whose blood genotype has been identified as BO;

Sample 6: the blood of a person whose blood genotype has been identified as OO.

1. Using the genomic DNA of the sample to be tested as a template, the primer combination prepared in step 1 is used for PCR amplification to obtain a PCR amplification product.

The reaction system consists of KCl, MgCl ₂ , bovine serum albumin, Tween-20, glycerol, NaN ₃ , dNTP, primer mix, Taq gold enzyme, template and Tris-HCl buffer; the primer mix is the primer combination prepared in step 1 A mixture of primers in . In the reaction system, the concentration of KCl is 50mM, the concentration of MgCl is _1.6mM , the concentration of bovine serum albumin is 0.8mg/mL, the concentration of Tween-20 is 0.2% (v/v), and the concentration of glycerol is 3.2% (v/v), the concentration of NaN ₃ is 0.02% (v/v), the concentration of dATP, dTTP, dGTP and dCTP is 200 μ M, primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO- The concentrations of R1, primer ABO-R2 and primer ABO-R3 are all 0.32μM, the concentration of Taq gold enzyme is 0.1U/μL, the template is 0.05ng/μL, the concentration of Tris-HCl buffer is pH8.3, 20mM Tris-HCl.

Reaction program: pre-denaturation at 95°C for 11 min; denaturation at 94°C for 30 s, annealing at 59°C for 2 min, extension at 72°C for 1 min, 28 cycles; extension at 60°C for 60 min; storage at 4°C.

2. After completing step 1, add 1 μL of PCR amplification product and 0.2 μL Typer500 internal standard to deionized formamide, and then dilute to 20 μL with deionized formamide to obtain a reaction solution.

3. After completing step 2, take the reaction solution, denature at 95°C for 5 minutes, quickly transfer to -20°C for 5 minutes, and then perform capillary electrophoresis detection with an ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 18s.

The experimental results are shown in Figure 1 (A is sample 1, B is sample 2, C is sample 3, D is sample 4, E is sample 5, F is sample 6). The results show that there are 4 alleles at the ABO blood type locus, which are named allele 1 (the size is 187bp, and the nucleotide sequence is shown in sequence 7 in the sequence table), allele 2 (the size is 190bp, the nuclear The nucleotide sequence is shown in sequence 8 in the sequence listing), allele 3 (the size is 192bp, and the nucleotide sequence is shown in sequence 9 in the sequence listing) and allele 4 (the size is 200bp, and the nucleotide sequence is as shown in Shown in sequence 10 in the sequence listing). 4 alleles correspond to 6 blood genotypes: sample 1 with blood type genotype AA contains allele 1 and allele 4; sample 2 with blood type genotype AB contains allele 1, allele 2 and allele 4; sample 3 with blood type genotype AO contains allele 1, allele 3 and allele 4; sample 4 with blood type genotype BB contains allele 2 and allele 4; Sample 5 with blood type genotype BO contains allele 1, allele 2, allele 3 and allele 4; sample 6 with blood type genotype OO contains allele 1 and allele 3.

Embodiment 2, the method for detecting ABO blood group genotype

1. Method for detecting ABO blood group genotype

The primer combination prepared in Step 1 of Example 1 can be used to detect ABO blood group genotype. The specific steps are: use the genomic DNA or total DNA of the sample to be tested as a template, use the primer combination prepared in step 1 to perform PCR amplification to obtain a PCR amplification product, and then determine the content of the PCR product according to the nucleotide sequence of the PCR amplification product Which of allele 1, allele 2, allele 3 and allele 4, and then make the following judgment: if allele 1 and allele 4 are contained, the ABO blood type genotype of the sample to be tested is AA; if it contains allele 1, allele 2 and allele 4, the ABO blood type genotype of the sample to be tested is AB; if it contains allele 1, allele 3 and allele 4, then The ABO blood type genotype of the sample to be tested is AO; if it contains allele 2 and allele 4, the ABO blood type genotype of the sample to be tested is BB; if it contains allele 1, allele 2, allele 3 and allele 4, the ABO blood genotype of the sample to be tested is BO; if it contains allele 1 and allele 3, the ABO blood genotype of the sample to be tested is OO.

2. Accuracy

1. Using 90 blood cards to be tested (round, with a diameter of 1.0mm) as templates, use the primers ABO-F1, primer ABO-F2, primer ABO-F3, and primer ABO-R1 prepared in step 1 of Example 1. , primer ABO-R2 and primer ABO-R3 for PCR amplification to obtain PCR amplification products.

The reaction system consists of KCl, MgCl ₂ , bovine serum albumin, Tween-20, glycerol, NaN ₃ , dNTP, primer mix, Taq gold enzyme, template and Tris-HCl buffer; the primer mix is composed of the above primers mixture. In the reaction system, the concentration of KCl is 50mM, the concentration of MgCl is _1.6mM , the concentration of bovine serum albumin is 0.8mg/mL, the concentration of Tween-20 is 0.2% (v/v), and the concentration of glycerol is 3.2% (v/v), the concentration of NaN ₃ is 0.02% (v/v), the concentration of dATP, dTTP, dGTP and dCTP is 200 μM, the concentration of each primer is 0.32 μM, and the concentration of Taq gold enzyme is 0.1 U/μL, the template is a blood card to be tested, the concentration of Tris-HCl buffer is pH8.3, 20mM Tris-HCl.

Reaction program: pre-denaturation at 95°C for 11 min; denaturation at 94°C for 30 s, annealing at 59°C for 2 min, extension at 72°C for 1 min, 28 cycles; extension at 60°C for 60 min; storage at 4°C.

2. After completing step 1, add 1 μL of PCR amplification product and 0.2 μL Typer500 internal standard to deionized formamide, and then dilute to 20 μL with deionized formamide to obtain a reaction solution.

3. After completing step 2, take the reaction solution, denature it at 95°C for 5 minutes, quickly transfer it to -20°C for 5 minutes, and then use the ABI3130xl genetic analyzer for capillary electrophoresis detection to obtain a DNA detection map; then determine the test according to the conclusion of step 1 The blood type genotype of the blood card. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 18s.

The blood genotypes of 90 blood cards to be tested were detected by classical serological methods.

The experimental results are shown in Table 1. The results show that the accuracy rate of ABO blood group genotype detection is 100% by adopting the method provided in the first step.

Table 1

Example 3, Preparation and verification of the template of the allelic typing standard of the ABO blood group locus

1. Obtaining recombinant plasmids

(1) Artificially synthesize DNA fragment A, DNA fragment B, DNA fragment C and DNA fragment D. The nucleotide sequence of DNA fragment A is shown as sequence 7 in the sequence listing. The nucleotide sequence of DNA fragment B is shown as sequence 8 in the sequence listing. The nucleotide sequence of DNA fragment C is shown as sequence 9 in the sequence listing. The nucleotide sequence of DNA fragment D is shown as sequence 10 in the sequence listing.

(2) Prepare 4 recombinant plasmids shown in Table 2, including 4 alleles of the ABO blood group loci in Example 1. According to the sequencing results, the detailed information of each recombinant plasmid is as follows:

Recombinant plasmid A: link the DNA fragment A synthesized in step (1) with plasmid pMD18-T to obtain recombinant plasmid A;

Recombinant plasmid B: link the DNA fragment B synthesized in step (1) with plasmid pMD18-T to obtain recombinant plasmid B;

Recombinant plasmid C: link the DNA fragment C synthesized in step (1) with plasmid pMD18-T to obtain recombinant plasmid C;

Recombinant plasmid D: Ligate the DNA fragment D synthesized in step (1) with plasmid pMD18-T to obtain recombinant plasmid D.

Table 2

serial number loci allele Recombinant plasmid name 1 ABO blood group loci 1 recombinant plasmid A 2 ABO blood group loci 2 recombinant plasmid B 3 ABO blood group loci 3 Recombinant plasmid C 4 ABO blood group loci 4 Recombinant plasmid D

2. Preparation of templates for allelic typing standards of ABO blood group loci

(1) Use Nanodrop 1000 micro-spectrophotometer to measure and dilute the concentration of recombinant plasmid DNA obtained in step 1 to 1 ng/μL to obtain dilutions of each recombinant plasmid.

(2) After completing step (1), take 1 μL of the dilutions of the recombinant plasmids and mix them, then dilute to 1 mL with ultrapure water to obtain the template of the allelic typing standards for ABO blood group loci.

In the template of the allelic typing standard of the ABO blood type locus, the DNA concentration of each allele in step 1 is 1 pg/μL.

3. Verification of the template of the allelic typing standard of the ABO blood group locus

(1) With the template of the allelic typing standard of the ABO blood type locus as a template, the primers ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 were used for PCR amplification to obtain PCR amplification products.

The reaction system and reaction procedure are the same as those in Step 2 of Example 1.

(2) After completing step (1), add 1 μL of PCR amplification product and 0.2 μL Typer500 internal standard to deionized formamide, and then dilute to 20 μL with deionized formamide to obtain a reaction solution.

(3) After step (2), the reaction solution was taken, denatured at 95°C for 5 minutes, quickly transferred to -20°C for 5 minutes, and then detected by capillary electrophoresis with an ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions are: the injection voltage is 1.2kV, and the injection time is 18s.

The experimental results are shown in Figure 2. The results showed that the allelic typing of the ABO blood group loci was complete and correct, with sharp and clear peak shapes, no miscellaneous peaks, and good balance. Therefore, the allelic ladder of the ABO blood group locus can be prepared using the template of the allelic ladder of the ABO blood group locus.

<110> Physical Evidence Identification Center of the Ministry of Public Security

<120> The method for detecting ABO blood group genotype and the template of the allelic typing standard of ABO blood group locus

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<170> PatentIn version 3.5

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Claims (10)

1. The primer combination is composed of primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3; The primer ABO-F1 is the following A1) or A2): A1) a single-stranded DNA molecule shown in Sequence 1 in the sequence listing; A2) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 1 and has the same function as Sequence 1; The primer ABO-F2 is as follows A3) or A4): A3) a single-stranded DNA molecule shown in Sequence 2 in the sequence listing; A4) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to Sequence 2 and has the same function as Sequence 2; The primer ABO-F3 is as follows A5) or A6): A5) a single-stranded DNA molecule shown in sequence 3 in the sequence listing; A6) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 3 and has the same function as sequence 3; The primer ABO-R1 is as follows A7) or A8): A7) a single-stranded DNA molecule shown in sequence 4 in the sequence listing; A8) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 4 and has the same function as sequence 4; The primer ABO-R2 is as follows A9) or A10): A9) the single-stranded DNA molecule shown in sequence 5 in the sequence listing; A10) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 5 and has the same function as sequence 5; The primer ABO-R3 is as follows A11) or A12): A11) the single-stranded DNA molecule shown in sequence 6 in the sequence listing; A12) A DNA molecule that undergoes one or several nucleotide substitutions and/or deletions and/or additions to sequence 6 and has the same function as sequence 6. 2. The application of the primer combination according to claim 1 is (b1) or (b2): (b1) preparing a test kit for detecting ABO blood group genotype; (b2) Detection of ABO blood group genotype. 3. A test kit containing the primer combination according to claim 1; said test kit is used to detect ABO blood group genotype. 4. The preparation method of the kit according to claim 3, comprising the step of packaging each primer separately. 5. A method for detecting ABO blood group genotype, comprising the steps of: taking the genomic DNA or total DNA of the sample to be tested as a template, using the primer combination described in claim 1 to carry out PCR amplification, obtaining the PCR amplification product, and then performing Judge as follows: (d1) If the PCR amplification product contains DNA fragment A and DNA fragment D, and does not contain DNA fragment B and DNA fragment C, the ABO blood type genotype of the sample to be tested is AA; (d2) If the PCR amplification product contains the DNA fragment A, the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment C, the ABO blood type genotype of the sample to be tested is AB; (d3) If the PCR amplification product contains the DNA fragment A, the DNA fragment C and the DNA fragment D, and does not contain the DNA fragment B, the ABO blood type genotype of the sample to be tested is AO; (d4) If the PCR amplification product contains the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment A and the DNA fragment C, the ABO blood type genotype of the sample to be tested is BB; (d5) If the PCR amplification product contains the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D, the ABO blood type genotype of the sample to be tested is BO; (d6) If the PCR amplification product contains the DNA fragment A and the DNA fragment C, and does not contain the DNA fragment B and the DNA fragment D, the ABO blood type genotype of the sample to be tested is OO; The nucleotide sequence of the DNA fragment A is shown in sequence 7 in the sequence listing; The nucleotide sequence of the DNA fragment B is shown in sequence 8 in the sequence listing; The nucleotide sequence of the DNA fragment C is shown in sequence 9 in the sequence listing; The nucleotide sequence of the DNA fragment D is shown as sequence 10 in the sequence listing. 6. A method for detecting ABO blood type genotype, comprising the steps of: detecting whether the genomic DNA or the total DNA of the sample to be tested contains DNA fragment A, DNA fragment B, DNA fragment C or DNA fragment D, and then judge as follows: (e1) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A and the DNA fragment D, and does not contain the DNA fragment B and the DNA fragment C, then the ABO blood group gene of the sample to be tested Type is AA; (e2) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment C, the ABO blood group gene of the sample to be tested Type is AB; (e3) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment C and the DNA fragment D, and does not contain the DNA fragment B, then the ABO blood group gene of the sample to be tested Type is AO; (e4) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment B and the DNA fragment D, and does not contain the DNA fragment A and the DNA fragment C, then the ABO blood group gene of the sample to be tested Type is BB; (e5) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D, the ABO blood type genotype of the sample to be tested is BO ; (e6) If the genomic DNA or total DNA of the sample to be tested contains the DNA fragment A and the DNA fragment C, and does not contain the DNA fragment B and the DNA fragment D, then the ABO blood group gene of the sample to be tested type is OO; The nucleotide sequence of the DNA fragment A is shown in sequence 7 in the sequence listing; The nucleotide sequence of the DNA fragment B is shown in sequence 8 in the sequence listing; The nucleotide sequence of the DNA fragment C is shown in sequence 9 in the sequence listing; The nucleotide sequence of the DNA fragment D is shown as sequence 10 in the sequence listing. 7. A DNA composition as a template of the allelic typing standard of the ABO blood type locus, containing DNA fragment A, DNA fragment B, DNA fragment C and DNA fragment D; The nucleotide sequence of the DNA fragment A is shown in sequence 7 in the sequence listing; The nucleotide sequence of the DNA fragment B is shown in sequence 8 in the sequence listing; The nucleotide sequence of the DNA fragment C is shown in sequence 9 in the sequence listing; The nucleotide sequence of the DNA fragment D is shown as sequence 10 in the sequence listing. 8. A recombinant plasmid composition as the template of the allelic typing standard of ABO blood type locus, it is characterized in that: described recombinant plasmid composition is prepared by following method: DNA fragment A, DNA fragment B , DNA fragment C and DNA fragment D are respectively inserted into 4 vectors to obtain 4 recombinant vectors; the 4 recombinant vectors are mixed to obtain a recombinant plasmid composition; The nucleotide sequence of the DNA fragment A is shown in sequence 7 in the sequence listing; The nucleotide sequence of the DNA fragment B is shown in sequence 8 in the sequence listing; The nucleotide sequence of the DNA fragment C is shown in sequence 9 in the sequence listing; The nucleotide sequence of the DNA fragment D is shown as sequence 10 in the sequence listing. 9. The recombinant plasmid composition according to claim 8, wherein the vector is a cloning vector. 10. The application of the DNA composition according to claim 7 or the recombinant plasmid composition according to claim 8 or 9 is a1) or a2) or a3): a1) as a template for the allelic typing standard of the ABO blood group locus; a2) preparing the allelic typing standards for ABO blood group loci; a3) Detecting the allelic typing of the ABO blood group loci.