CN108660198B - PCR-SBT method and reagent for genotyping of platelet membrane protein CD36 antigen - Google Patents
PCR-SBT method and reagent for genotyping of platelet membrane protein CD36 antigen Download PDFInfo
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Abstract
The invention provides a PCR-SBT method for typing CD36 antigen, which comprises the following steps of preparing human genome DNA; providing amplification primers, and respectively amplifying the gene sequences of the CD36 antigen by using polymerase chain reaction; carrying out double enzyme digestion purification on the amplification product; providing a sequencing primer, and carrying out sequencing PCR reaction on the purified product; purifying the sequencing product by a sodium acetate-ethanol precipitation method, and performing capillary electrophoresis sequencing; the obtained sequence was analyzed by software to determine its genotype. The invention also provides a reagent used in the method. The invention obtains the CD36 antigen gene typing oligonucleotide sequence by respectively sequencing the gene sequences of the 5' end and 1-15 exons of the CD36 antigen, and precisely types the gene. The invention has important practical significance for medical research units, pharmaceutical research units and reagent development units.
Description
Technical Field
The invention relates to a genotyping detection method, in particular to a molecular biology detection method for a human platelet membrane protein CD36 deletion mutant gene, and a reagent applied to the method.
Background
In recent years, blood transfusion has become one of the important means for treating thrombocytopenia in clinical blood collection and platelet transfusion due to the development of blood transfusion technology. However, some chronic, long-term transfused patients experience the appearance of ineffective platelet infusions as the number of infusions increases. Therefore, the invalidation of platelet transfusion becomes a very troublesome problem in clinic, and causes wide attention in the transfusion world. The reasons for the ineffectiveness of platelet infusion are divided into non-immune factors and immune factors, and in recent years, post-infusion complications caused by the deletion of CD36 antigen in immune factors have become a research hotspot. In response to this, we developed a sequencing kit to detect the gene mutation point of the CD36 antigen deletion and speculated the relationship between CD36 antigen deletion and platelet infusion ineffectiveness. The detection of the CD36 antigen is expected to be a routine test item in transfusion laboratories, thereby reducing the incidence of complications after platelet transfusion.
The human CD36 gene has a full length of 32Kb, is located on chromosome 7q.2, and contains 15 exons. Exons 1, 2 and 15 are non-coding regions, the remaining 12 exons are involved in coding amino acids, and exons 3 and 14 encode the N and C termini of a CD36 protein molecule. There are independent first exons and 3 independent promoters on the CD36 gene, with no TATA box sequences and CpG islands. The research on the mutation of the CD36 gene has been reported in a large number of documents. More than 20 CD36 mutant genes have been detected to date, resulting in CD36 antigen deletions. The gene mutation mechanism comprises single nucleotide substitution, base deletion, base insertion and exon skipping in mRNA splicing processing, so that 1 or a plurality of exons in the mature mRNA generated by transformation are deleted. Gene mutations can produce two types of CD36 antigen deletions. Individuals with type 1 CD36 loss do not express CD36 antigen on the platelet and monocyte surfaces, and individuals with type 2 loss do not express CD36 antigen only on the platelets. Form II is more common than form I. Platelet CD36 loss was very rare in caucasians (0.3%), and was highly frequent in asian (3-10%) and african populations (7%). Recently, CD36 (-) platelet hoof selection has been reported for German blood donors, and no negative specimens have been found so far. The gene mutation found in association with the deletion of CD36 is significantly different in african populations and african U.S. populations. The CD36 gene is highly polymorphic. The CD36 gene of an individual in the African western refractory disease region is researched to discover 24 mutants.
The epitope on platelet CD36 is called Naka, and its corresponding antibody is designated anti-Naka antibody. anti-Naka was first identified in patients who had platelet transfusion null (PTR) after receiving multiple HLA isotype platelet transfusions. A number of cases have since reported that anti-Naka antibodies are closely related to transfusion reactions. anti-Naka antibodies can also cause immune thrombocytopenia, early fetal death (repeat early death), and other diseases. Nakajima et al (2008) have demonstrated in recent studies that anti-Naka plays a key role in the development of transfusion-associated acute lung injury (TRALI).
At present, the domestic research on CD36 focuses on the association of the CD36 with lipid metabolism and glucose metabolism diseases, and the systematic research on the CD36 whole gene polymorphism and the research on blood transfusion safety are lacked. The loss of CD36 is characterized by a higher frequency than the loss of other glycoproteins of platelets, and individuals with CD36 deficiency often do not exhibit symptoms, which undoubtedly increases the incidence of transfusion adverse reactions in CD 36-deficient patients. To date, it has been found that CD36 gene variation is associated with a variety of diseases such as metabolic syndrome, lactose intolerance, insulin resistance, myocardial infarction, atherosclerosis, coronary heart disease, diabetes, hypertension, hyperlipidemia, angiogenesis, thrombotic diseases, stroke, alzheimer's disease, and rejection of disease.
The current methods for identifying CD36 mainly comprise serological methods and genotyping methods aiming at the transfusion risk possibly caused by the loss of platelet CD 36. The serological methods for identifying granulocyte antigens or antibodies mainly include methods such as granulocyte agglutination test, granulocyte immunofluorescence test (GIFT), Monoclonal Antibody-specific granulocyte antigen capture test (Monoclonal Antibody immunization Of granular antigen, MAIGA), flow cytometry, ELISA and the like. The gene typing method of granulocyte antigen system mainly includes PCR-RFLP, PCR-SSP, PCR-SSO, etc. Bux et al showed that the genotyping method was as reliable as the MAIGA method for typing CD36, whereas GIFT had a 15% typing error rate. The current main method for genotyping the CD36 antigen is PCR-SSP (PCR-sequence specific primer), which needs multi-tube amplification, and the PCR-SSP method can only be designed for some specific sites with distinctiveness when designing primers, so that only conventional CD36 antigen can be genotyped, and some special new mutation sites are difficult to be defined. The PCR-SBT (PCR-Sequence Based Typing) Typing method of the CD36 can overcome the defects and limitations and is the most accurate Typing method. However, the current PCR-SBT typing method for CD36 antigen gene is not complete enough, and although there are also laboratories adopting PCR-SBT method to genotype CD36 antigen, the same PCR condition and dry PCR primer of CD36 gene have not been systematic sequenced on PCR plate so far. Therefore, the establishment of a PCR-SBT method of the human platelet membrane protein CD36 deletion mutant gene has important significance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a PCR-SBT method of human platelet membrane protein CD36 deletion type mutant gene, so as to overcome the defects in the existing genotyping technology. Therefore, the invention adopts the following technical scheme
It carries out sequence sequencing gene typing on the gene sequences of the 5' end and 1-15 exons of the CD36 antigen respectively, and comprises the following steps:
(1) providing an amplification primer, drying the primer by using a centrifugal dryer, placing the primer at the bottom of a detachable 96-hole PCR plate, sealing a membrane, and storing at-20 ℃ for later use;
(2) preparing human genome DNA;
(3) respectively amplifying gene sequences of 5' end and 1-15 exons of CD36 gene in human genome DNA by polymerase chain reaction;
(4) carrying out double enzyme digestion purification on the amplification product obtained in the step (3);
(5) providing a sequencing primer, and carrying out sequencing PCR reaction on the purified product obtained in the step (4);
(6) purifying the sequencing product obtained in the step (5) by a sodium acetate-ethanol precipitation method, and performing capillary electrophoresis sequencing;
(7) and (4) analyzing the sequence obtained in the step (6) by software to determine the genotype of the sequence. The two enzymes required for purification in the step (4) are shrimp alkaline phosphatase and exonuclease I.
Another technical problem to be solved by the present invention is to provide reagents for use in the above method. For this purpose, the invention adopts the following technical scheme that the kit consists of a primer for amplification and an oligonucleotide sequencing primer for sequencing analysis; the primers used for amplification were:
CD36-5'-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5'-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA
the sequences of the 2 oligonucleotide sequencing primers are as follows:
M13F:TGTAAAACGACGGCCAGT
M13R :CAGGAAACAGCTATGACC
in addition, CD36-EXON1 adds 2 sequencing primers:
Ploy-TF:GCTGTGTGGGGGATTTTTTTTTT
Ploy-TR:AGAGAAGAGAAAGCACTC。
the design of the primers in the invention is the key of PCR amplification, and the method and software related to the design of the primers can be freely obtained from the Internet. The oligonucleotide primer designed by the invention is designed and obtained according to a continuous oligonucleotide sequence including polymorphic sites in a human CD36 antigen gene sequence in GenBank. The amplification primers for the gene sequence of the CD36 antigen system were designed based on the sequence numbered NC-000007.13 (GI:224589819) in GenBank. All the forward amplification primers are connected with 16 base sequences TGTAAAACGACGGCCAGT on the forward sequence of the M13 vector at the 5 'ends, all the reverse amplification primers are connected with 16 base sequences CAGGAAACAGCTATGACC on the reverse sequence of the M13 vector at the 5' ends, all the forward amplification primers and the reverse amplification primers respectively have common adaptor sequences after connection, and then the adaptor sequences are selected as sequencing primers. The invention uses 16 pairs of oligonucleotide primers to respectively amplify 16 gene segments of the CD36 antigen, and can ensure the effective amplification of CD36 antigen system genes. The design of the amplification primer avoids the polymorphic site of the CD36 antigen coding sequence and avoids the omission of any mutation point. The design of the sequencing primer can ensure that the sequences of the amplified fragments can be clearly and accurately measured, and the sequences are subjected to bidirectional sequencing, so that the sample is subjected to accurate gene typing. The invention obtains the oligonucleotide sequence of the CD36 gene by respectively sequencing the gene sequences of the 5' end and 1-15 exons of the CD36 antigen, and accurately shapes the gene. With the popularization of DNA sequence analyzers, the PCR-SBT technology is widely applied to clinical detection. The application of the coding sequence information of all CD36 antigen systems obtained at high throughput in aspects of gene typing, gene polymorphism detection, gene frequency investigation and analysis and the like is widely regarded. The reagent and the method provided by the invention can be used as an independent and widely-applied identification method, solve the problem that 16 fragments of the gene sequences of the CD 365' end and the 1-15 exons obtain accurate sequences, exert the characteristics of high throughput and accurate result of the operation of PCR-SBT on CD36 gene typing, are highly valued in the fields of clinical blood transfusion medical research, genetics and the like, and have important practical significance for medical research units, pharmaceutical research and reagent development units. Particularly, the CD36 antigen system distribution of the blood donated people is determined, and the blood containing the CD36 antibody is prevented from being infused, so that the transfusion adverse reaction generated by the granulocyte antibody is prevented, and the safety of the blood is improved.
Drawings
FIG. 1 shows a detachable 96-well PCR plate according to the present invention. Wherein, the number of the rows is 1 to 12, each row is 8 holes, and the two rows are one person, and the total number of the holes is 16. The PCR plate can be split between two columns. Columns 1 and 2 are one part, 3, 4 are another part, and so on, 6 parts are a plate.
FIG. 2 is a schematic diagram of a detachable PCR plate without cutting or cutting. The figure shows a PCR plate without primers.
FIG. 3 is a PCR amplification electropherogram of the CD36 antigen gene detected from the specimen of the invention. I is an amplified fragment of CD 36-5', 2 is an amplified fragment of CD36-EXON1, 3 is an amplified fragment of CD36-EXON2, 4 is an amplified fragment of CD36-EXON3, 5 is an amplified fragment of CD36-EXON4, 6 is an amplified fragment of CD36-EXON5, 7 is an amplified fragment of CD36-EXON6, 8 is an amplified fragment of CD36-EXON7, 9 is an amplified fragment of CD36-EXON8, 10 is an amplified fragment of CD36-EXON9, 11 is an amplified fragment of CD36-EXON10, 12 is an amplified fragment of CD36-EXON11, 13 is an amplified fragment of CD36-EXON12, 14 is an amplified fragment of CD36-EXON13, 15 is a amplified fragment of CD36-EXON14, and EX16 is an amplified fragment of CD36-EXON 15.
FIG. 4 is a partial sequencing electropherogram of the invention detecting 3 sample CD 36. A, G, C, T shows the four bases sequenced, A is adenine, G is guanine, C is cytosine and T is thymine.
Detailed description of the preferred embodiments
The present invention will be described in further detail with reference to examples.
Example I
The present invention is described in detail by taking the CD36 antigen genotyping of blood donors as an example, and the PCR-SBT method for CD36 antigen genotyping adopted by the present invention specifically comprises the following steps
I. Human genomic DNA was prepared as a template for PCR amplification in the subsequent steps. Taking 400ml of whole blood to be detected, and obtaining the whole blood according to Magcore
The HF16 autosampler instructions extract genomic DNA and determine concentration. .
2. 16 pairs of amplification primers and 4 sequencing primers are synthesized, the specific sequences are shown in the sequence in the content of the invention, the details are not repeated, the amplification primers are diluted to 10 mu M by using pure water, the primers, phenol red (100 mu g/ml) and the pure water are dried and placed at the bottom of a detachable 96-hole PCR plate by using a centrifugal dryer according to the table 1, and the primers, the phenol red and the pure water are stored at the temperature of minus 20 ℃ for later use after membrane sealing.
Table 1 CD36 primer configuration.
3. PCR Buffer configuration (360. mu.l): 10 Xbuffer (Lot: AC6901A, TaKaRa), dNTP (Lot: BL3018, TaKaRa), ReadyPCR (Lot: G9RP106, inno-train) and purified water were prepared as described in Table 2, and stored at-20 ℃ until use.
Table 2 PCR Buffer configuration.
4. Preparing r-Taq enzyme (Lot: AGY0124A, TaKaRa), PCR amplification template prepared in step I and PCR amplification template prepared in step 3, preparing a PCR amplification system according to the system described in Table 3, and adding 25 mu l of the PCR amplification system into the PCR plate prepared in step 2 (16 wells/person part in total) after uniformly mixing.
Table 3 PCR amplification system for CD36 antigen.
In Table 3, 400. mu.l of the total system was amplified by a PCR apparatus (ABI, Vertiri) according to the following procedure at 95 ℃ for 5min, and the DNA double strand was sufficiently disentangled; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, binding the primer to the template, extending at 72 ℃ for 1min, extending the required amplified fragment, and reacting for 35 cycles; the amplified fragment was fully extended at 72 ℃ for 10 min.
5. And (5) double enzyme digestion purification of the amplification product. And detecting the amplified fragments of the sample, and performing agarose gel electrophoresis on5 mu l of PCR products respectively, as shown in figure 3, so as to determine the specificity of the amplified fragments. Mu.l of a mixture of Shrimp Alkaline Phosphatase (SAP) and exonuclease I (Exo-I), Exo-SAP (lot: 160904, TBG), was added to 10. mu.l of the remaining PCR product, and the amplification product was purified by using the nucleotide Y-terminal dephosphorylation function of Shrimp Alkaline Phosphatase (SAP) and the single strand-specific Y-exonuclease function of exonuclease I (Exo-I). Mu.l of Exo-SAP was added to 10. mu.l of the amplification product system, and the enzyme digestion was carried out at 37 ℃ for 30min and at 80 ℃ for 15 min.
6. Sequencing PCR was performed on the PCR product. And (3) adding 75ml of pure water into the PCR product purified in the step 3, and uniformly mixing. The two sequencing primers described in the summary of the invention were diluted with pure water to a concentration of 3.2 μmol/L, and a reaction system was prepared according to Table 4 using BigDyeterminator V3.1 sequencing Kit (ABI, USA), wherein the sequencing primer I was any one of M13F, M13R, Ploy-TF and Ploy-TR. The samples were diluted 1:4 to obtain amplified and purified fragments as templates, and then subjected to 4 reactions of M13F, M13R, Ploy-TF and Ploy-TR, respectively, and 2 reactions of M13F and M13R, respectively, except CD36-EXON 1. Amplifying the pre-denaturation at 96 ℃ for 2min by using a PCR instrument (ABI, VerritiL) according to the following program, and fully untying the DNA double strand; denaturation at 96 deg.C for 10s, annealing at 50 deg.C for 5s, binding sequencing primer to DNA template, extension at 60 deg.C for 4min, 29 cycles, and extension of amplified fragment at 72 deg.C for 5 min.
Table 4 PCR sequencing system of PCR products in step 5.
7. The sequencing amplification PCR product is directly purified by a sodium acetate/ethanol purification method. And (4) directly purifying the sequencing amplification PCR product in the step (4) by using a sodium acetate/ethanol purification method. Adding mixed solution of I mul EDTA I, 25 mu m and 1 mul sodium acetate (3 mu m)/absolute ethyl alcohol (25ml) into the PCR product directly, mixing uniformly, centrifuging for 30min at 2000g, removing supernatant, adding 80ml 70% ethyl alcohol, centrifuging for 10min at 2000g, removing supernatant, adding 10 mul formamide after the ethyl alcohol is volatilized, dissolving, denaturing at 95 ℃ for 5min, and cooling on ice rapidly.
8. And (3) carrying out 16-hole capillary high-throughput electrophoresis sequencing on the prepared product on an ABI 3130XL sequencer, carrying out sequence comparison on the sequencing result by utilizing Seqmann 7.0 software, determining the genotype of the CD36 antigen system, and displaying a partial sequence of the CD36 gene of the detection sample. Wherein, FIG. 3 is the PCR amplification electrophoresis pattern of the antigen gene of the detection specimen of the invention. 1 is an amplified fragment of CD 36-5', 2 is an amplified fragment of CD36-EXON1, 3 is an amplified fragment of CD36-EXON2, 4 is an amplified fragment of CD36-EXON3, 5 is an amplified fragment of CD36-EXON4, 6 is an amplified fragment of CD36-EXON5, 7 is an amplified fragment of CD36-EXON6, 8 is an amplified fragment of CD36-EXON7, 9 is an amplified fragment of CD36-EXON8, 10 is an amplified fragment of CD36-EXON9, 11 is an amplified fragment of CD36-EXON10, 12 is an amplified fragment of CD36-EXON11, 13 is an amplified fragment of CD36-EXON12, 14 is an amplified fragment of CD36-EXON13, 15 is an amplified fragment of CD36-EXON14, and EX16 is an amplified fragment of CD36-EXON 15.
FIG. 4 is a partial sequencing electropherogram of the CD36 gene of 3 samples detected by the present invention. A, G, C, T shows the four bases sequenced, A is adenine, G is guanine, C is cytosine and T is thymine. Sample 1 is CD36 exon5 deletion two bases 329-330 del AC; sample 2 is CD36 with one base A mutated into T in exon 12; sample 3 was CD36 exon13 deleted 12 bases 1228-1239 del ATTGTGCCTATT.
In conclusion, the reagent and the method provided by the invention can be used as an independent and widely-applied identification method, solve the problem of accurate typing identification of the CD36 gene, exert the characteristics of accurate typing result and high-throughput operation of the CD36 gene by PCR-SBT, have high importance on relevant applications in the fields of clinical transfusion medical research, genetics and the like, and have important significance on medical research units, pharmaceutical research and reagent development units. Especially, the distribution of the granulocyte antigen system of the blood donation people is determined, the blood containing granulocyte antibodies is prevented from being infused, so that the transfusion adverse reaction generated by the granulocyte antibodies is prevented, the TRALI is effectively prevented, and the safety of the blood is improved.
Sequence listing
<110> Guangzhou blood center
<120> PCR-SBT method and reagent for platelet membrane protein CD36 antigen genotyping
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<213> Artificial Sequence (Artificial Sequence)
<400> 19
tgtaaaacga cggccagtat ggactacact ggaggag 37
<210> 20
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
caggaaacag ctatgacctt ggaagatgca gaagaaca 38
<210> 21
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgtaaaacga cggccagttt catgcttggc tattgagtt 39
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
caggaaacag ctatgacctc tttcttctgc cctaat 36
<210> 23
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgtaaaacga cggccagtgc ctgaaagctt tacatattg 39
<210> 24
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
caggaaacag ctatgacccc ataggaagaa atcgacc 37
<210> 25
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgtaaaacga cggccagtaa ccttgacatt cgattgg 37
<210> 26
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
caggaaacag ctatgaccga gatgctatca aatgctca 38
<210> 27
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tgtaaaacga cggccagtta tttcagttcc ccgaga 36
<210> 28
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
caggaaacag ctatgacctt tgttcaattg gatcat 36
<210> 29
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgtaaaacga cggccagtct gatgactaac accaatagag 40
<210> 30
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
caggaaacag ctatgacctg gacaactttg gcacaa 36
<210> 31
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgtaaaacga cggccagtca tcatttccac aactg 35
<210> 32
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
caggaaacag ctatgaccat tagcctagaa caaagtggta 40
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
tgtaaaacga cggccagt 18
<210> 34
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
caggaaacag ctatgacc 18
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gctgtgtggg ggattttttt ttt 23
<210> 36
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
agagaagaga aagcactc 18
Claims (2)
1. A PCR-SBT primer group for the genotyping of platelet membrane protein CD36 antigen is characterized by consisting of a primer for amplification and an oligonucleotide sequencing primer for sequencing analysis; the primer for amplification is an amplification primer of a gene sequence of the CD36 antigen, and the sequence is as follows:
CD36-5'-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5'-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA；
the oligonucleotide sequencing primer sequences are as follows:
M13F : TGTAAAACGACGGCCAGT
M13R : CAGGAAACAGCTATGACC。
2. the use of the primer set of claim 1 for preparing a PCR-SBT reagent for genotyping the platelet membrane protein CD36 antigen, characterized in that the use comprises the following steps:
(1) providing an amplification primer, drying the primer by using a centrifugal dryer, placing the primer at the bottom of a detachable 96-hole PCR plate, sealing a membrane, and storing at-20 ℃ for later use;
(2) preparing human genome DNA;
(3) respectively amplifying the gene sequences of the 5' end of the CD36 antigen and the 1-15 exons in the human genome DNA by using polymerase chain reaction;
(4) carrying out double enzyme digestion purification on the amplification product obtained in the step (3);
(5) providing a sequencing primer, and carrying out sequencing PCR reaction on the purified product obtained in the step (4);
(6) purifying the sequencing product obtained in the step (5) by a sodium acetate-ethanol precipitation method, and performing capillary electrophoresis sequencing;
(7) analyzing the sequence obtained in the step (6) by software to determine the genotype of the sequence;
the amplification primers in the step (1) are 16 pairs of oligonucleotides to respectively amplify the gene sequences of the 5' end of the CD36 antigen and the 1-15 exons,
the amplification primer sequences of the gene sequence of the CD36 antigen are as follows:
CD36-5’-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA
CD36-5’-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG
CD36-EXON1-F TGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG
CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC
CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC
CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTATT
CD36-EXON3-F TGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA
CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG
CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC
CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG
CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT
CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG
CD36-EXON6-F TGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG
CD36-EXON6-R CAGGAAACAGCTATGACCAATTATGCCTTGCCAATGC
CD36-EXON7-F TGTAAAACGACGGCCAGTCCTCACCTCAACATAGTAAGA
CD36-EXON7-R CAGGAAACAGCTATGACCGAGTTAATACCTAGCAGAACAG
CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC
CD36-EXON8-R CAGGAAACAGCTATGACCCTCTGAATCATGCAGTAAGGG
CD36-EXON9-F TGTAAAACGACGGCCAGTATGGACTACACTGGAGGAG
CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA
CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT
CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT
CD36-EXON11-F TGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG
CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC
CD36-EXON12-F TGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG
CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA
CD36-EXON13-F TGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA
CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT
CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG
CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA
CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG
CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA；
the sequencing primer in the step (5) is 2 oligonucleotide sequencing primers, and the sequence is as follows:
M13F：TGTAAAACGACGGCCAGT，
M13R：CAGGAAACAGCTATGACC；
in addition, CD36-EXON1 adds 2 sequencing primers:
Ploy-TF:GCTGTGTGGGGGATTTTTTTTTT
Ploy-TR:AGAGAAGAGAAAGCACTC；
the two enzymes required for purification in the step (4) are shrimp alkaline phosphatase and exonuclease I.
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