CN106834483B - Method for detecting ABO blood group genotype and template of allelic typing standard of ABO blood group locus - Google Patents
Method for detecting ABO blood group genotype and template of allelic typing standard of ABO blood group locus Download PDFInfo
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Abstract
本发明公开了检测ABO血型基因型的方法和ABO血型基因座的等位基因分型标准物的模板。本发明所提供的方法，包括如下步骤：以待测样本的基因组DNA或总DNA为模板，采用引物组合进行PCR扩增，得到PCR扩增产物，然后确定含有哪几种等位基因，进一步确定待测样本的ABO血型基因型。引物组合由6条引物组成，核苷酸序列依次为序列表中的序列1至序列6。实验证明，采用本发明提供的方法检测ABO血型基因型，准确率为100％；采用本发明提供的重组质粒组合物作为ABO血型基因座的等位基因分型标准物的模板，可扩增获得ABO血型基因座的等位基因分型标准物，并可在多种商业试剂盒中使用。本发明具有重要的应用价值。The invention discloses a method for detecting ABO blood type genotype and a template for an allele typing standard of ABO blood type locus. The method provided by the present invention includes the following steps: taking the genomic DNA or total DNA of the sample to be tested as a template, using a primer combination to carry out PCR amplification to obtain a PCR amplification product, then determining which alleles are contained, and further determining The ABO blood type genotype of the sample to be tested. The primer combination consists of 6 primers, and the nucleotide sequences are sequence 1 to sequence 6 in the sequence listing. Experiments show that the method provided by the present invention is used to detect the ABO blood group genotype, and the accuracy rate is 100%; the recombinant plasmid composition provided by the present invention is used as the template of the allelic typing standard of the ABO blood group locus, which can be amplified and obtained Allelic typing standards for the ABO blood group locus and are available in a variety of commercial kits. The invention has important application value.
Description
Technical Field
The invention relates to the technical field of forensic medicine, in particular to a method for detecting ABO blood group genotypes and a template of an allele typing standard substance of an ABO blood group locus.
Background
The ABO blood type is a classical human genetic marker and plays an important role in the fields of blood transfusion, paternity test, anthropology research, forensic material evidence inspection and the like. For the ABO blood group, the conventional serological test method is to test antigen or antibody, and the most common method in the forensic physical evidence test is to test antigen substance, but since the ABO blood group antigen is glycolipid or glycoprotein, the test is susceptible to factors such as antigen activity, antibody specificity, and microbial contamination. Particularly aiming at the mixed spot inspection in the sexual crime, the traditional serological inspection method also has the insurmountable limitation, so that the blood group substances of female victims and male suspects cannot be separated, the blood group of the suspects can be presumed according to the blood group of the victims, and once the blood group of the male substance in the mixed spot is shielded by the blood group of the victims, the blood group of the male substance in the mixed spot cannot be accurately deduced. In 1985, the Gill research group applies a differential lysis method and a DNA fingerprint map technology to forensic testing to successfully extract sperm DNA and female DNA in the mixed spots respectively, solves the problem of puzzling forensic physical evidence testing for years, and makes it possible for the DNA technology to test the sperm blood type in the mixed spots. In 1990, Yamamoto et al reported nucleotide sequences of ABO blood group genes, accurately detected differences of different SNP site bases of each allele on DNA sequences, and these studies confirmed that genotyping of ABO blood group by using differences of SNP sites on ABO blood group genes would be a very effective method, particularly for trace or degraded samples in forensic practice, but existing SNP typing of ABO blood group genes still has some defects, such as unrepeatability; although the method can be repeated, the peak pattern on the capillary electrophoresis platform is easy to have the phenomena of bifurcation, shoulder peak, asymmetry and the like, so that the typing can not be accurately read.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect the ABO blood type and genotype.
In order to solve the above technical problems, the present invention provides a primer combination.
The primer combination provided by the invention can be composed of a primer ABO-F1, a primer ABO-F2, a primer ABO-F3, a primer ABO-R1, a primer ABO-R2 and a primer ABO-R3;
the primer ABO-F1 can be A1) or A2) as follows:
A1) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
A2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the primer ABO-F2 can be A3) or A4) as follows:
A3) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
A4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer ABO-F3 can be A5) or A6) as follows:
A5) a single-stranded DNA molecule shown as a sequence 3 in a sequence table;
A6) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;
the primer ABO-R1 can be A7) or A8) as follows:
A7) a single-stranded DNA molecule shown as a sequence 4 in a sequence table;
A8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the primer ABO-R2 can be A9) or A10) as follows:
A9) a single-stranded DNA molecule shown as a sequence 5 in a sequence table;
A10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the primer ABO-R3 can be A11) or A12) as follows:
A11) a single-stranded DNA molecule shown as a sequence 6 in a sequence table;
A12) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 6 and has the same function as the sequence 6.
The application of the primer combination also belongs to the protection scope of the invention. The application of the primer combination can be (b1) or (b 2):
(b1) preparing a kit for detecting ABO blood group genotype;
(b2) and detecting the ABO blood type and gene type.
The kit containing the primer combination also belongs to the protection scope of the invention. The kit can be used for detecting ABO blood group genotypes.
The preparation method of the kit containing the primer combination also belongs to the protection scope of the invention. The preparation method of the kit containing the primer combination can comprise the step of packaging each primer separately.
In order to solve the technical problems, the invention also provides a method for detecting the ABO blood type and the genotype.
The method for detecting the ABO blood group genotype provided by the invention can be specifically the method one, and comprises the following steps: taking the genome DNA or the total DNA of a sample to be detected as a template, carrying out PCR amplification by adopting the primer combination to obtain a PCR amplification product, and then judging as follows:
(d1) if the PCR amplification product contains a DNA fragment A and a DNA fragment D, and does not contain a DNA fragment B and a DNA fragment C, the ABO blood type genotype of the sample to be detected 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 detected 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 detected 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 detected 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 detected 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 detected is OO;
the nucleotide sequence of the DNA fragment A is shown as a sequence 7 in a sequence table;
the nucleotide sequence of the DNA fragment B is shown as a sequence 8 in a sequence table;
the nucleotide sequence of the DNA fragment C is shown as a sequence 9 in a sequence table;
the nucleotide sequence of the DNA fragment D is shown as a sequence 10 in a sequence table.
In the above method, the reaction system for performing PCR amplification may be KCl or MgCl2Bovine serum albumin, Tween-20, glycerol and NaN3dNTP, a primer mixture, Taq gold medal enzyme, a template and a Tris-HCl buffer solution; the primer mixture is a mixture consisting of all primers in the primer combination. In the reaction system, the concentration of KCl may be specifically 50mM, MgCl2The concentration of (b) may be 1.6mM, Bovine Serum Albumin (BSA) may be 0.8mg/mL, Tween-20 may be 0.2% (v/v), glycerol (glycerol) may be 3.2% (v/v), and NaN3The concentration of (b) may be 0.02% (v/v), the concentrations of dATP, dTTP, dGTP and dCTP may be 200. mu.M, the concentrations of the primer ABO-F1, the primer ABO-F2, the primer ABO-F3, the primer ABO-R1, the primer ABO-R2 and the primer ABO-R3 may be 0.32. mu.M, the concentration of Taq gold-brand enzyme may be 0.1U/. mu.L, the concentration of the template may be 0.05 ng/. mu.L, and the concentration of Tris-HCl buffer may be Tris-HCl, pH8.3 and 20 mM.
In the above method, the reaction procedure of "performing PCR amplification" may specifically be: pre-denaturation at 95 ℃ for 11 min; denaturation at 94 deg.C for 30s, annealing at 59 deg.C for 2min, extension at 72 deg.C for 1min, and 28 times of circulation; extending for 60min at 60 ℃; storing at 4 ℃.
The method for detecting the ABO blood group genotype provided by the invention can be specifically a method II, and comprises the following steps: detecting whether the genome DNA or the total DNA of a sample to be detected contains the DNA fragment A, the DNA fragment B, the DNA fragment C or the DNA fragment D, and then judging as follows:
(e1) if the genome DNA or the total DNA of the sample to be detected contains the DNA fragment A and the DNA fragment D, and does not contain the DNA fragment B and the DNA fragment C, the ABO blood type and the genotype of the sample to be detected are AA;
(e2) if the genome DNA or the total DNA of the sample to be detected 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 detected is AB;
(e3) if the genome DNA or the total DNA of the sample to be detected 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 and the genotype of the sample to be detected are AO;
(e4) if the genome DNA or the total DNA of the sample to be detected 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 and the genotype of the sample to be detected are BB;
(e5) if the genome DNA or the total DNA of the sample to be detected contains the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D, the ABO blood type and the genotype of the sample to be detected are BO;
(e6) and if the genome DNA or the total DNA of the sample to be detected 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 detected is OO.
In order to solve the above technical problems, the present invention also provides a DNA composition as a template for an allelic typing standard of an 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 may be 1: (0.8-1.2): (0.8-1.2): (0.8 to 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 technical problems, the invention also provides a recombinant plasmid composition used as a template of an allelic typing standard of the ABO blood group locus. The recombinant plasmid composition is prepared by the following method: inserting the DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D into 4 vectors respectively 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 may specifically be plasmid pMD 18-T.
The application of the DNA composition or the recombinant plasmid composition also belongs to the protection scope of the invention. The application of the DNA composition or the recombinant plasmid composition is a1) or a2) or a 3):
a1) as a template for an allelic typing standard for an ABO blood group locus;
a2) preparing an allelic typing standard of an ABO blood group locus;
a3) detecting allelic typing of the ABO blood group locus.
The DNA fragment A, the DNA fragment B, the DNA fragment C and the DNA fragment D can all be obtained by taking human genome DNA as a template and carrying out PCR amplification by adopting the primer combination (each primer is not subjected to fluorescent modification).
The 5' ends of the primer ABO-F1, the primer ABO-F2 and the primer ABO-F3 can be subjected to fluorescence modification so as to facilitate capillary electrophoresis detection of PCR amplification products. The 5' ends of the primer ABO-F1, the primer ABO-F2 and the primer ABO-F3 can be modified by TAMRA.
Experiments prove that the ABO blood type and genotype are detected by adopting the primer combination provided by the invention, and the accuracy rate is 100%; the recombinant plasmid composition provided by the invention is used as a template of the allele typing standard substance of the ABO blood group locus, can be amplified to obtain the allele typing standard substance of the ABO blood group locus, and can be used in various commercial kits. The invention has important application value.
Drawings
FIG. 1 shows the results of the second step of example 1.
FIG. 2 shows the experimental results of step 3 of example 3.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Typer500 internal standard is a product of a public security department material evidence authentication center. The deionized formamide is a product of ABI company, and has a catalog number of 4311320. ABI3130xl genetic Analyzer and Nanodrop1000 Microspectrophotometer are both products of ABI. The plasmid pMD18-T is a product of TaKaRa, Cat 6011.
Example 1 detection of the genotype of alleles of the ABO blood group Locus
Preparation of primer combination
The primer combinations used to detect the genotype of the alleles of the ABO blood group locus are as follows:
primer ABO-F1: 5 ' -TAMRA-ACACCCCGGAAGGATGTCCTCGTGGTGA-3 ' (TAMRA modification is carried out at the 5 ' end) (sequence 1 in a sequence table);
primer ABO-F2: 5 ' -TAMRA-GGAAGGATGTCCTCGTGGTA-3 ' (TAMRA modification is carried out at the 5 ' end) (sequence 2 in a sequence table);
primer ABO-F3: 5 ' -TAMRA-AGTGGACGTGGACATGGAGTTCC-3 ' (TAMRA modification is carried out at the 5 ' end) (sequence 3 in a sequence table);
primer ABO-R1: 5'-AATGTCCACAGTCACTCGCCACT-3' (SEQ ID NO: 4 in the sequence Listing);
primer ABO-R2: 5'-CCCGAAGAACCCCCCCAG-3' (SEQ ID NO: 5 in the sequence Listing);
primer ABO-R3: 5'-TTTTTCGAAGAACGCCCCCAT-3' (SEQ ID NO: 6 in the sequence Listing).
Primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 were artificially synthesized.
Secondly, detecting the genotype of allele of ABO blood group locus
The sample to be detected is sample one, sample two, sample three, sample four, sample five or sample six:
a first sample: blood of a human having a blood group genotype of AA already identified;
sample two: blood of a person having been assigned a blood group genotype of AB;
sample three: human blood having been characterized as having a blood group genotype of AO;
sample four: blood of a human having been identified with a blood group genotype of BB;
sample five: blood of a human having been assigned a blood group genotype of BO;
sample six: blood of a person having been identified with a blood group genotype of OO.
1. And (3) performing PCR amplification by using the genome DNA of the sample to be detected as a template and adopting the primer combination prepared in the step one to obtain a PCR amplification product.
The reaction system consists of KCl and MgCl2Bovine serum albumin, Tween-20, glycerol and NaN3dNTP, a primer mixture, Taq gold medal enzyme, a template and a Tris-HCl buffer solution; the primer mixture is the mixture composed of the primers in the primer combination prepared in the first step. The concentration of KCl in the reaction system is 50mM, and MgCl2The concentration of (a) 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 GlycineThe oil concentration was 3.2% (v/v), NaN3The concentration of (2) was 0.02% (v/v), the concentrations of dATP, dTTP, dGTP and dCTP were all 200. mu.M, the concentrations of primer ABO-F1, primer ABO-F2, primer ABO-F3, primer ABO-R1, primer ABO-R2 and primer ABO-R3 were all 0.32. mu.M, the concentration of Taq gold-brand enzyme was 0.1U/. mu.L, the template was 0.05 ng/. mu.L, and the concentration of Tris-HCl buffer was Tris-HCl pH8.3, 20 mM.
Reaction procedure: pre-denaturation at 95 ℃ for 11 min; denaturation at 94 deg.C for 30s, annealing at 59 deg.C for 2min, extension at 72 deg.C for 1min, and 28 times of circulation; extending for 60min at 60 ℃; storing at 4 ℃.
2. After step 1 was completed, 1. mu.L of PCR amplification product and 0.2. mu.L of Typer500 internal standard were added to deionized formamide, and then the volume was made 20. mu.L with deionized formamide to obtain a reaction solution.
3. And (3) after the step 2 is finished, taking the reaction solution, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ and standing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 18 s.
The experimental results are shown in fig. 1(a is sample one, B is sample two, C is sample three, D is sample four, E is sample five, and F is sample six). The result shows that the ABO blood type locus has 4 alleles which are respectively named as allele 1 (the size is 187bp, the nucleotide sequence is shown as sequence 7 in the sequence table), allele 2 (the size is 190bp, the nucleotide sequence is shown as sequence 8 in the sequence table), allele 3 (the size is 192bp, the nucleotide sequence is shown as sequence 9 in the sequence table) and allele 4 (the size is 200bp, the nucleotide sequence is shown as sequence 10 in the sequence table). 4 alleles correspond to 6 blood group genotypes: a sample I with the blood group genotype of AA contains an allele 1 and an allele 4; a sample II with the blood group genotype AB contains allele 1, allele 2 and allele 4; a sample III with the blood group genotype of AO contains allele 1, allele 3 and allele 4; a sample four with the blood group genotype BB contains an allele 2 and an allele 4; the sample five with the blood group genotype BO contains allele 1, allele 2, allele 3 and allele 4; sample six, with the blood group genotype OO, contains allele 1 and allele 3.
Example 2 method for detecting ABO blood group genotype
Method for detecting ABO blood type and genotype
ABO blood group genotype can be detected by using the primer combination prepared in the first step of example 1. The method comprises the following specific steps: taking genome DNA or total DNA of a sample to be detected as a template, performing PCR amplification by adopting the primer combination prepared in the step one to obtain a PCR amplification product, determining which one of allele 1, allele 2, allele 3 and allele 4 is contained according to the nucleotide sequence of the PCR amplification product, and then judging as follows: if the sample contains the allele 1 and the allele 4, the ABO blood type genotype of the sample to be detected is AA; if the sample contains allele 1, allele 2 and allele 4, the ABO blood type genotype of the sample to be detected is AB; if the sample contains allele 1, allele 3 and allele 4, the ABO blood type genotype of the sample to be detected is AO; if the sample contains the allele 2 and the allele 4, the ABO blood type genotype of the sample to be detected is BB; if the sample contains allele 1, allele 2, allele 3 and allele 4, the ABO blood type genotype of the sample to be detected is BO; if the sample contains allele 1 and allele 3, the ABO blood group genotype of the sample to be tested is OO.
Second, accuracy
1. Respectively taking 90 blood cards to be detected (circular, with the diameter of 1.0mm) as templates, and carrying out PCR amplification by using the primer ABO-F1, the primer ABO-F2, the primer ABO-F3, the primer ABO-R1, the primer ABO-R2 and the primer ABO-R3 prepared in the step one in the embodiment 1 to obtain PCR amplification products.
The reaction system consists of KCl and MgCl2Bovine serum albumin, Tween-20, glycerol and NaN3dNTP, a primer mixture, Taq gold medal enzyme, a template and a Tris-HCl buffer solution; the primer mixture is the mixture composed of the above primers. The concentration of KCl in the reaction system is 50mM, and MgCl2The concentration of (A) is 1.6mM, the concentration of bovine serum albumin is 0.8mg/mL, the concentration of Tween-20 is 0.2% (v/v), the concentration of glycerol is 3.2% (v/v), and NaN3The concentration of (b) is 0.02% (v/v), and the concentrations of dATP, dTTP, dGTP and dCTP are all200 mu M, the concentration of each primer is 0.32 mu M, the concentration of Taq gold medal enzyme is 0.1U/mu L, the template is 1 blood card to be detected, and the concentration of Tris-HCl buffer solution is Tris-HCl with pH8.3 and 20 mM.
Reaction procedure: pre-denaturation at 95 ℃ for 11 min; denaturation at 94 deg.C for 30s, annealing at 59 deg.C for 2min, extension at 72 deg.C for 1min, and 28 times of circulation; extending for 60min at 60 ℃; storing at 4 ℃.
2. After step 1 was completed, 1. mu.L of PCR amplification product and 0.2. mu.L of Typer500 internal standard were added to deionized formamide, and then the volume was made 20. mu.L with deionized formamide to obtain a reaction solution.
3. After the step 2 is finished, taking the reaction solution, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ and standing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a DNA detection map; and then determining the blood type and the genotype of the blood card to be detected according to the conclusion of the step one. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 18 s.
And (3) detecting the blood type and the genotype of 90 blood cards to be detected by adopting a classical serological method.
The results are shown in Table 1. The result shows that the method provided by the first step is adopted to detect the ABO blood type and the genotype, and the accuracy rate is 100%.
TABLE 1
Example 3 preparation and validation of templates for allelic typing standards for ABO blood group loci
1. Obtaining of recombinant plasmid
(1) Artificially synthesizing a DNA fragment A, a DNA fragment B, a DNA fragment C and a DNA fragment D. The nucleotide sequence of the DNA fragment A is shown as a sequence 7 in a sequence table. The nucleotide sequence of the DNA fragment B is shown as a sequence 8 in the sequence table. The nucleotide sequence of the DNA fragment C is shown as a sequence 9 in a sequence table. The nucleotide sequence of the DNA fragment D is shown as a sequence 10 in a sequence table.
(2) 4 recombinant plasmids shown in Table 2 were prepared containing 4 alleles of the ABO blood group locus in example 1. According to the sequencing results, the details of each recombinant plasmid are as follows:
recombinant plasmid a: connecting the DNA fragment A synthesized in the step (1) with a plasmid pMD18-T to obtain a recombinant plasmid A;
recombinant plasmid B: connecting the DNA fragment B synthesized in the step (1) with a plasmid pMD18-T to obtain a recombinant plasmid B;
recombinant plasmid d: connecting the DNA fragment C synthesized in the step (1) with a plasmid pMD18-T to obtain a recombinant plasmid C;
recombinant plasmid D: and (2) connecting the DNA fragment D synthesized in the step (1) with a plasmid pMD18-T to obtain a recombinant plasmid D.
TABLE 2
| Numbering | Genetic loci | Alleles | Name of recombinant plasmid |
| 1 | ABO blood group loci | 1 | |
| 2 | ABO |
2 | |
| 3 | ABO |
3 | Recombinant plasmid C |
| 4 | ABO blood group loci | 4 | Recombinant plasmid D |
2. Preparation of templates for allelic typing standards for ABO blood group loci
(1) The concentration of the recombinant plasmid DNA obtained in step 1 was measured and diluted to 1 ng/. mu.L using a Nanodrop1000 microspectrophotometer, and dilutions of the respective recombinant plasmids were obtained.
(2) And (3) after the step (1) is finished, mixing 1 mu L of diluent of each recombinant plasmid, and then using ultrapure water to perform constant volume to 1mL to obtain a template of the allele typing standard substance of the ABO blood type locus.
In the template of the allelic typing standard for the ABO blood group locus, the DNA concentration of each allele in the first step was 1 pg/. mu.L.
3. Validation of templates for allelic typing standards for ABO blood group loci
(1) And (3) performing PCR amplification by using a template of an allele typing standard of an ABO blood group locus as a template and adopting the primer ABO-F1, the primer ABO-F2, the primer ABO-F3, the primer ABO-R1, the primer ABO-R2 and the primer ABO-R3 prepared in the step one in the example 1 to obtain a PCR amplification product.
The reaction system and the reaction procedure were the same as in step two 1 of example 1.
(2) After the completion of the step (1), 1. mu.L of the PCR amplification product and 0.2. mu.L of the Typer500 internal standard were added to deionized formamide, and then the volume was made to 20. mu.L with deionized formamide to obtain a reaction solution.
(3) And (3) after the step (2) is finished, taking the reaction solution, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ and standing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 18 s.
The results of the experiment are shown in FIG. 2. The result shows that all alleles of the ABO blood type gene locus have complete and correct typing, sharp and clear peak types, no miscellaneous peaks and good balance. Thus, an allelic typing standard for an ABO blood group locus can be prepared using a template for an allelic typing standard for an ABO blood group locus.
<110> material evidence identification center of public security department
<120> method for detecting ABO blood group genotype and template of allele typing standard of ABO blood group locus
<160>10
<170>PatentIn version 3.5
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| 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 |
| CN112708699B (en) * | 2020-03-13 | 2023-11-03 | 苏州白垩纪生物科技有限公司 | Virus sample inactivation and cracking 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 |
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