CN108660198B

CN108660198B - A PCR-SBT method and reagent for platelet membrane protein CD36 antigen genotyping

Info

Publication number: CN108660198B
Application number: CN201810460870.5A
Authority: CN
Inventors: 丁浩强; 付涌水; 叶欣
Original assignee: GUANGZHOU BLOOD CENTER
Current assignee: GUANGZHOU BLOOD CENTER
Priority date: 2018-05-15
Filing date: 2018-05-15
Publication date: 2022-02-22
Anticipated expiration: 2038-05-15
Also published as: CN108660198A

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

PCR-SBT method and reagent for genotyping of platelet membrane protein CD36 antigen

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

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgtaaaacga cggccagtaa aataagtttc gcaagctca 39

<210> 2

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caggaaacag ctatgacctc ccccacacag cacattactg 40

<210> 3

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgtaaaacga cggccagtgc tgaatatctc agatatagg 39

<210> 4

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caggaaacag ctatgaccgt atttatacag tagtgtcacc 40

<210> 5

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgtaaaacga cggccagtcc tgtagtctat ccaaagtc 38

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caggaaacag ctatgaccgt atggtaacag atgttttatt 40

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgtaaaacga cggccagtgt aggcattaga agcaagaa 38

<210> 8

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caggaaacag ctatgaccag tcgcatcata tagagttg 38

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgtaaaacga cggccagtaa agcgtcactc taaagc 36

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggaaacag ctatgaccat gacatttgcc aagtagaag 39

<210> 11

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgtaaaacga cggccagtct atctggcata ttctgtgt 38

<210> 12

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caggaaacag ctatgaccaa gcatcttcct gtaatctg 38

<210> 13

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtaaaacga cggccagtgg aatgtcgtct tcttgtg 37

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggaaacag ctatgaccaa ttatgccttg ccaatgc 37

<210> 15

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgtaaaacga cggccagtcc tcacctcaac atagtaaga 39

<210> 16

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caggaaacag ctatgaccga gttaatacct agcagaacag 40

<210> 17

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgtaaaacga cggccagttg atctggctac ctaatggc 38

<210> 18

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

caggaaacag ctatgaccct ctgaatcatg cagtaaggg 39

<210> 19

<211> 37

<212> DNA

<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

1. a PCR-SBT primer set for platelet membrane protein CD36 antigen genotyping, characterized in that, it is made up of primers for amplification and oligonucleotide sequencing primers for sequencing analysis; said primers for amplification The primers are the amplification primers for the gene sequence of the CD36 antigen, and the sequence is as follows:

CD36-5'-F TGTAAAACGACGGCCAGT AAAATAAGTTTCGCAAGCTCA

CD36-5'-R CAGGAAACAGCTATGACC TCCCCCACACAGCACATTACTG

CD36-EXON1-F TGTAAAACGACGGCCAGT GCTGAATATCTCAGATATAGG

CD36-EXON1-R CAGGAAACAGCTATGACC GTATTTATACAGTAGTGTCACC

CD36-EXON2-F TGTAAAACGACGGCCAGT CCTGTAGTCTATCCAAAGTC

CD36-EXON2-R CAGGAAACAGCTATGACC GTATGGTAACAGATGTTTTTATT

CD36-EXON3-F TGTAAAACGACGGCCAGT GTAGGCATTAGAAGCAAGAA

CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG

CD36-EXON4-F TGTAAAACGACGGCCAGT AAAGCGTCACTCTAAAGC

CD36-EXON4-R CAGGAAACAGCTATGACC ATGACATTTGCCAAGTAGAAG

CD36-EXON5- FTGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT

CD36-EXON5-R CAGGAAACAGCTATGACC AAGCATCTTCCTGTAATCTG

CD36-EXON6-F TGTAAAACGACGGCCAGT GGAATGTCGTCTTCTTGTG

CD36-EXON6-R CAGGAAACAGCTATGACC AATTATGCCTTGCCAATGC

CD36-EXON7-F TGTAAAACGACGGCCAGT CCTCACCTCAACATAGTAAGA

CD36-EXON7-R CAGGAAACAGCTATGACC GAGTTAATACCTAGCAGAACAG

CD36-EXON8-F TGTAAAACGACGGCCAGTTGATCTGGCTACCTAATGGC

CD36-EXON8-R CAGGAAACAGCTATGACC CTCTGAATCATGCAGTAAGGG

CD36-EXON9-F TGTAAAACGACGGCCAGT ATGGACTACACTGGAGGAG

CD36-EXON9-R CAGGAAACAGCTATGACCTTGGAAGATGCAGAAGAACA

CD36-EXON10-F TGTAAAACGACGGCCAGTTTCATGCTTGGCTATTGAGTT

CD36-EXON10-R CAGGAAACAGCTATGACCTCTTTCTTCTGCCCTAAT

CD36-EXON11-F TGTAAAACGACGGCCAGT GCCTGAAAGCTTTACATATTG

CD36-EXON11-R CAGGAAACAGCTATGACC CCATAGGAAGAAATCGACC

CD36-EXON12-F TGTAAAACGACGGCCAGT AACCTTGACATTCGATTGG

CD36-EXON12-R CAGGAAACAGCTATGACC GAGATGCTATCAAATGCTCA

CD36-EXON13-F TGTAAAACGACGGCCAGT TATTTCAGTTCCCCGAGA

CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT

CD36-EXON14-F TGTAAAACGACGGCCAGT CTGATGACTAACACCAATAGAG

CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA

CD36-EXON15-F TGTAAAACGACGGCCAGT CATCATTTCCACAACTG

CD36-EXON15-R CAGGAAACAGCTATGACC ATTAGCCTAGAACAAAGTGGTA;

The oligonucleotide sequencing primer sequences are as follows:

M13F : TGTAAAACGACGGCCAGT

M13R: CAGGAAACAGCTATGACC.

2. the application of primer set described in claim 1 in the PCR-SBT reagent that prepares platelet membrane protein CD36 antigen genotyping, it is characterized in that described application comprises the following steps:

(1) Provide amplification primers, use a centrifugal dryer to dry the primers and place them on the bottom of a detachable 96-well PCR plate, and store them at -20°C after sealing;

(2) preparation of human genomic DNA;

(3) Amplify the gene sequences of CD36 antigen 5' end and 1-15 exons in human genomic DNA respectively by polymerase chain reaction;

(4) double-enzymatic digestion and purification of the amplified product obtained in step (3);

(5) providing sequencing primers, and subjecting the purified product obtained in step (4) to a sequencing PCR reaction;

(6) purifying the sequencing product obtained in step (5) by sodium acetate-ethanol precipitation, and performing capillary electrophoresis sequencing;

(7) the sequence obtained in step (6) is analyzed by software to determine its genotype;

The amplification primers in the step (1) are 16 pairs of oligonucleotides to respectively amplify the gene sequences of the CD36 antigen 5' end and exons 1-15,

The amplification primer sequences of the gene sequence of CD36 antigen are as follows:

CD36-5’-F TGTAAAACGACGGCCAGTAAAATAAGTTTCGCAAGCTCA

CD36-5’-R CAGGAAACAGCTATGACCTCCCCCACACAGCACATTACTG

CD36-EXON1-FTGTAAAACGACGGCCAGTGCTGAATATCTCAGATATAGG

CD36-EXON1-R CAGGAAACAGCTATGACCGTATTTATACAGTAGTGTCACC

CD36-EXON2-F TGTAAAACGACGGCCAGTCCTGTAGTCTATCCAAAGTC

CD36-EXON2-R CAGGAAACAGCTATGACCGTATGGTAACAGATGTTTTTATT

CD36-EXON3-FTGTAAAACGACGGCCAGTGTAGGCATTAGAAGCAAGAA

CD36-EXON3-R CAGGAAACAGCTATGACCAGTCGCATCATATAGAGTTG

CD36-EXON4-F TGTAAAACGACGGCCAGTAAAGCGTCACTCTAAAGC

CD36-EXON4-R CAGGAAACAGCTATGACCATGACATTTGCCAAGTAGAAG

CD36-EXON5-F TGTAAAACGACGGCCAGTCTATCTGGCATATTCTGTGT

CD36-EXON5-R CAGGAAACAGCTATGACCAAGCATCTTCCTGTAATCTG

CD36-EXON6-FTGTAAAACGACGGCCAGTGGAATGTCGTCTTCTTGTG

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-FTGTAAAACGACGGCCAGTGCCTGAAAGCTTTACATATTG

CD36-EXON11-R CAGGAAACAGCTATGACCCCATAGGAAGAAATCGACC

CD36-EXON12-FTGTAAAACGACGGCCAGTAACCTTGACATTCGATTGG

CD36-EXON12-R CAGGAAACAGCTATGACCGAGATGCTATCAAATGCTCA

CD36-EXON13-FTGTAAAACGACGGCCAGTTATTTCAGTTCCCCGAGA

CD36-EXON13-R CAGGAAACAGCTATGACCTTTGTTCAATTGGATCAT

CD36-EXON14-F TGTAAAACGACGGCCAGTCTGATGACTAACACCAATAGAG

CD36-EXON14-R CAGGAAACAGCTATGACCTGGACAACTTTGGCACAA

CD36-EXON15-F TGTAAAACGACGGCCAGTCATCATTTCCACAACTG

CD36-EXON15-R CAGGAAACAGCTATGACCATTAGCCTAGAACAAAGTGGTA;

The sequencing primers in the step (5) are 2 oligonucleotide sequencing primers, and the sequences are 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.