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Method for detecting ABO blood-group genotype and template for allelic ladder of ABO blood-group gene locus

An ABO blood type and genotype technology, applied in the field of forensic science, can solve problems such as asymmetry, inability to repeat, and inability to accurately read typing

Active Publication Date: 2017-06-13
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

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

Method used

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  • Method for detecting ABO blood-group genotype and template for allelic ladder of ABO blood-group gene locus
  • Method for detecting ABO blood-group genotype and template for allelic ladder of ABO blood-group gene locus
  • Method for detecting ABO blood-group genotype and template for allelic ladder of ABO blood-group gene locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, detect the genotype of the allele of ABO blood type locus

[0070] 1. Preparation of primer combinations

[0071] The primer combinations for detecting the genotypes of the alleles of the ABO blood group locus are as follows:

[0072] Primer ABO-F1: 5'-TAMRA-ACACCCCGGAAGGATGTCCTCGTGGTGA-3' (TAMRA modification at the 5' end) (sequence 1 in the sequence listing);

[0073] Primer ABO-F2: 5'-TAMRA-GGAAGGATGTCCTCGTGGTA-3' (TAMRA modification at the 5' end) (sequence 2 in the sequence listing);

[0074] Primer ABO-F3: 5'-TAMRA-AGTGGACGTGGACATGGAGTTCC-3' (TAMRA modification at the 5' end) (sequence 3 in the sequence listing);

[0075] Primer ABO-R1: 5'-AATGTCCACAGTCACTCGCCACT-3' (sequence 4 in the sequence listing);

[0076] Primer ABO-R2: 5'-CCCGAAGAACCCCCCCAG-3' (sequence 5 in the sequence listing);

[0077] Primer ABO-R3: 5'-TTTTTCGAAGAACGCCCCCAT-3' (sequence 6 in the sequence listing).

[0078] The primers ABO-F1, ABO-F2, ABO-F3, ABO-R1, ABO-R2 and ABO-R...

Embodiment 2

[0093] Embodiment 2, the method for detecting ABO blood group genotype

[0094] 1. Method for detecting ABO blood group genotype

[0095]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;...

Embodiment 3

[0106] Example 3, Preparation and verification of the template of the allelic typing standard of the ABO blood group locus

[0107] 1. Obtaining recombinant plasmids

[0108] (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.

[0109] (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:

[0110] Recombinant plasmid A: link the DNA fragment A synthesized in step (1) with plasmid pMD18-T to obtain recombinant pla...

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Abstract

The invention discloses a method for detecting the ABO blood-group genotype and a template for an allelic ladder of an ABO blood-group gene locus. The method comprises the following steps: taking a genome DNA or general DNA of a to-be-detected sample as a template to perform PCR amplification by adopting primer combination to obtain a PCR amplified product; then, determining which alleles are contained, and further determining the ABO blood-group genotype of the to-be-detected sample, wherein the primer combination consists of 6 primers having nucleotide sequences being sequence 1 to sequence 6 in the sequence table sequentially. Experiments prove that the precision rate for detecting the ABO blood-group genotype by adopting the method is 100 percent; the recombinant plasmid composition provided by the method, serving as a template of an allelic ladder of an ABO blood-group gene locus, can be amplified to obtain the allelic ladder of the ABO blood-group gene locus, and can be applied to various commercial kits. The method and the template have an important application value.

Description

technical field [0001] 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 [0002] 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 impossi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/63
CPCC12Q1/6876C12Q2600/156C12Q2600/166
Inventor 王乐叶健赵兴春季安全孙启凡白雪马温华陈曼
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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