WO2017028408A1

WO2017028408A1 - Group-specific amplification primer, sequence-based typing method, and kit for hla genes

Info

Publication number: WO2017028408A1
Application number: PCT/CN2015/097065
Authority: WO
Inventors: 于浩洋; 徐筠娉; 蒂拉努斯·马塞尔·G·J
Original assignee: 深圳市晋百慧生物有限公司
Priority date: 2015-08-19
Filing date: 2015-12-10
Publication date: 2017-02-23
Also published as: CN105039332B; CN105039332A

Abstract

The present invention provides a group-specific amplification primer, sequence-based typing method, and kit for HLA genes. A sequence of the PCR amplification primer is selected from a sequence represented by SEQ ID NO.1-28, a sequence represented by SEQ ID NO.29-64, and a sequence represented by SEQ ID NO.65-94. By means of the present invention, a group-specific amplification primer is designed by performing serum classification on HLA genes, so as to perform sequence-based typing on all allelic genes of HLA-A, -B, and -C, and perform amplification sequencing on full-length sequences of HLA-A, -B, and -C.

Description

Group-specific amplification primers, sequencing typing methods and kits for HLA genes

Technical field

The present invention relates to biomedical and immunogenetics, in particular to group-specific amplification primers, sequencing typing methods and kits for HLA genes.

Background technique

The human leukocyte antigen system (HLA) is the most diverse and important immune genetic system in the human genome, and its diversity determines the body's resistance to infectious diseases, autoimmune diseases, genetic diseases and various tumors. In clinical applications, the degree of matching of genetic polymorphisms between donors and recipients directly determines the survival rate of hematopoietic stem cell transplantation and organ transplant patients.

In the HLA organization industry, there are three short-board problems in the domestic and international markets: First, most products are only classified for some exons of the HLA gene, such as only HLA-I genes (including Two exons of 2, 3, and 4 of HLA-A, -B, and -C) were tested for typing, but in fact, HLA-I full-length genes contained a total of seven exons, seven introns, and At both ends of the regulatory area, the absolute high score results have a total of 8 named names, such as A*02:01:01:01, and the current domestic and foreign reagents can only determine the first 4 results, so if HLA single nucleotide polymorphism In the region other than the exon detected by the reagent, it is easy to produce ambiguous results (Ambiguities). The current HLA classification products are ambiguous and can be as high as 40%-60%. Secondly, the PCR amplification primers for HLA typing products on the market are designed to be versatile, that is, the same site uses the same pair of amplification primers to amplify the HLA gene sequence of all populations, due to the allele of HLA gene. The number of genes is large. As of January 2015, the number of HLA-A, -B, and -C alleles in the world is 2995, 3760, and 2553, respectively (release 3.19.0, http://www.ebi. Ac.uk/ipd/imgt/hla), according to the serological classification of 28, 60 and 10, respectively, the sequence of alleles contained in each serologist line is quite different, giving the site a general expansion The design of the primers brings a lot of trouble. Even if it is barely designed, it is easy to produce allele dropouts of some family alleles. Atria has also reported that this phenomenon exists in its HLA sequencing products. Finally, the traditional HLA sequencing typing technique is to amplify the paternal parental diploid. Due to the high HLA diversity, the extremely dense heterozygous peak increases the difficulty and time of software development and results analysis.

CN 101962676 A discloses a human leukocyte antigen HLA-A, B gene full-length sequence determination and HLA gene sequencing typing method, and the HLA-A and -B gene full-length sequence determination methods include: a, each pair of primer pairs The full-length sequence of about 4 kb of HLA-A and B genes was amplified by PCR; b. The amplified product was cloned into pGEM-Tea sy body, and the full-length sequence was detected by the positive and negative bi-directional sequencing primers. A full-length sequence of 4.3 kb of the 38 alleles of HLA-A and a full length of 3.7 kb of the 30 alleles of HLA-B were obtained. It is also the use of primers with versatility of the site, which is likely to cause missed detection of some family alleles.

Summary of the invention

The invention aims to solve the three short board problems in the above-mentioned domestic and international HLA high-resolution sequencing typing technology, solve the defects of the existing HLA sequencing typing products, and achieve the HLA absolute high score of the tested specimen, and the invention provides a kind of Group-specific amplification primers, sequencing typing methods and kits for HLA genes.

To achieve the object of the present invention, the present invention adopts the following technical solutions:

In a first aspect, the invention provides a group-specific amplification primer for an HLA gene, the PCR amplification primer sequence being selected from the group consisting of the sequences set forth in SEQ ID NO. 1-28, SEQ ID NO. 29-64 Sequence and the sequence shown in SEQ ID NO. 65-94.

According to the group-specific classification of HLA-A, -B, and -C genes, the full-length haplotype-specific amplification primers are designed to amplify HLA-A, -B, and -C genes; After the addition, a universal forward and reverse full-length sequencing primer was designed for each gene to measure all exons and introns of HLA gene. sequence.

Preferably, the sequence shown in SEQ ID NO. 1-28 is a primer sequence for amplifying the HLA-A gene.

Preferably, the sequence set forth in SEQ ID NO. 1-2 is for alleles of the HLA-A* and A*36 sublines.

Preferably, the sequences set forth in SEQ ID NO. 3-4 are all alleles used to amplify the HLA-A*02, A*68, A*69 and A*80 sublines.

Preferably, the sequences set forth in SEQ ID NO. 5-6 are all alleles used to amplify the HLA-A*02 and A*69 sublines.

Preferably, the sequences set forth in SEQ ID NO. 7-8 are all alleles used to amplify the HLA-A*01, A*36, A*68, A*69 and A*80 sublines.

Preferably, the sequences set forth in SEQ ID NO. 9-10 are all alleles used to amplify the HLA-A*23 and A*24 sublines.

Preferably, the sequences set forth in SEQ ID NO. 11-12 are all alleles used to amplify the HLA-A*31 and A*33 sublines.

Preferably, the sequences set forth in SEQ ID NO. 13-14 are all alleles used to amplify the HLA-A*29, A*32 and A*74 sublines.

Preferably, the sequences set forth in SEQ ID NO. 15-16 are all alleles used to amplify the HLA-A*03 and A*30 sublines.

Preferably, the sequences set forth in SEQ ID NO. 17-18 are used to amplify HLA-A*01, A*02, A*03, A*11, A*36, A*68, A*69. And all alleles of the A*80 subfamily.

Preferably, the sequences set forth in SEQ ID NO. 19-20 are all alleles used to amplify the HLA-A*30 subline.

Preferably, the sequences set forth in SEQ ID NO. 21-22 are all alleles used to amplify the HLA-A*11 subline.

Preferably, the sequences set forth in SEQ ID NO. 23-24 are all alleles used to amplify the HLA-A*29 and A*80 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 25-26 are used to amplify HLA-A*25, A*26, A*29, A*31, A*32, A*33, A*34. All alleles of the A*66, A*74 and A*43 sublines.

Preferably, the sequence set forth in SEQ ID NO. 27-28 is an allele used to amplify all serologist lines of HLA-A as a positive control. .

In the present invention, the above-described group-specific PCR amplification primer sequences of the HLA-A gene are shown in Table 1.

Table 1

Note: “F” and “R” in the primer name indicate the upstream and downstream of the primer, respectively.

Preferably, the sequence set forth in SEQ ID NO. 29-64 is a primer sequence for amplifying the HLA-B gene.

Preferably, the sequences set forth in SEQ ID NO. 29-30 are all alleles used to amplify the HLA-B*07 and B*48 sublines.

Preferably, the sequences set forth in SEQ ID NO. 31-32 are all alleles used to amplify the HLA-B*08 subline.

Preferably, the sequence set forth in SEQ ID NO. 33-34 is for amplifying the HLA-B*13 subline All alleles.

Preferably, the sequences set forth in SEQ ID NO. 35-36 are all alleles used to amplify the HLA-B*15 and B*46 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 37-38 are all alleles used to amplify the HLA-B*14, B*38, B*39 and B*67 sublines.

Preferably, the sequences set forth in SEQ ID NO. 39-40 are all alleles used to amplify the HLA-B*15, B*51 and B*52 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 41-42 are all alleles used to amplify the HLA-B*35 and B*73 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 43-44 are all alleles used to amplify the HLA-B*18, B*44, B*73 and B*82 sublines.

Preferably, the sequences set forth in SEQ ID NO. 45-46 are all alleles used to amplify the HLA-B*07, B*40:01 and B*81 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 47-48 are all alleles used to amplify the HLA-B*18, B*27, B*37 and B*40:02/06 sublines.

Preferably, the sequences set forth in SEQ ID NO. 49-50 are all alleles used to amplify the HLA-B*41, B*45, B*49 and B*50 sublines.

Preferably, the sequences set forth in SEQ ID NO. 51-52 are all alleles used to amplify the HLA-B*42 subline.

Preferably, the sequences set forth in SEQ ID NOS. 53-54 are all alleles used to amplify the HLA-B*54, B*55, B*56 and B*59 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 55-56 are all alleles used to amplify the HLA-B*57 subline.

Preferably, the sequences set forth in SEQ ID NOS. 57-58 are used to amplify HLA-B*18, B*27, B*35, B*37, B*40, B*41, B*45 All alleles of the B*49, B*50, B*51, B*52, B*53, B*58 and B*78 sublines.

Preferably, the sequences set forth in SEQ ID NO. 59-60 are for amplifying all of the HLA-B*07, B*08, B*15, B*42, B*44 and B*47 sublines. Allele.

Preferably, the sequences shown in SEQ ID NO. 61-62 are used to amplify HLA-B*41, B*45, B*49, B*50, B*51, B*52, B*53. All alleles of the B*58 and B*78 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 63-64 are alleles used to amplify HLA-B* all serologist lines as a positive control.

In the present invention, the above-described group-specific PCR amplification primer sequences of the HLA-B gene are shown in Table 2.

Table 2

Preferably, the sequence set forth in SEQ ID NO. 65-94 is a primer sequence for amplifying the HLA-C gene.

Preferably, the sequences set forth in SEQ ID NO. 65-66 are all alleles used to amplify the HLA-C*01 and C*02 sublines.

Preferably, the sequences set forth in SEQ ID NO. 67-68 are all alleles used to amplify the HLA-C*01 subline.

Preferably, the sequences set forth in SEQ ID NO. 69-70 are all alleles used to amplify the HLA-C*02 subline.

Preferably, the sequences set forth in SEQ ID NO. 71-72 are all alleles used to amplify the HLA-C*03 subline.

Preferably, the sequences set forth in SEQ ID NOS. 73-74 are all alleles used to amplify the HLA-C*03 subline.

Preferably, the sequences set forth in SEQ ID NOS. 75-76 are all alleles used to amplify the HLA-C*04 and C*18 sublines.

Preferably, the sequences set forth in SEQ ID NO. 77-78 are all alleles used to amplify the HLA-C*05 and C*08 sublines.

Preferably, the sequences set forth in SEQ ID NO. 79-80 are used to amplify HLA-C*06 and C*12 All alleles of the subfamily.

Preferably, the sequences set forth in SEQ ID NOS. 81-82 are all alleles used to amplify the HLA-C*07 and C*18 sublines.

Preferably, the sequences set forth in SEQ ID NOS. 83-84 are all alleles used to amplify the HLA-C*07 subline.

Preferably, the sequences set forth in SEQ ID NOS. 85-86 are all alleles used to amplify the HLA-C*14 subline.

Preferably, the sequences set forth in SEQ ID NOS. 87-88 are all alleles used to amplify the HLA-C*15 subline.

Preferably, the sequences set forth in SEQ ID NO. 89-90 are all alleles used to amplify the HLA-C*16 subline.

Preferably, the sequences set forth in SEQ ID NO. 91-92 are all alleles used to amplify the HLA-C*17 subline.

Preferably, the sequences set forth in SEQ ID NO. 93-94 are alleles used to amplify all serologist lines of HLA-C as a positive control.

In the present invention, the above-described group-specific PCR amplification primer sequences of the HLA-C gene are shown in Table 3.

table 3

Preferably, the product fragment obtained by PCR amplification of the PCR amplification primer comprises all exons, introns and regulatory regions of the two ends of the corresponding HLA-A, -B and -C genes.

In a second aspect, the present invention provides a full-length sequencing primer for a gene of HLA-A, -B and -C amplified using a PCR amplification primer according to the first aspect, characterized in that the gene is The sequence of the full-length sequencing primer is selected from the group consisting of the sequences set forth in SEQ ID NO. 95-108, the sequence set forth in SEQ ID NO. 109-122, and the sequence set forth in SEQ ID NO. 123-136.

Preferably, the sequences set forth in SEQ ID NO. 95-108 are used to sequence the full length of the HLA-A gene.

In the present invention, the full-length sequencing primer sequences of the above HLA-A gene are shown in Table 4.

Table 4

Preferably, the sequence set forth in SEQ ID NO. 109-122 is used to sequence the full length of the HLA-B gene.

In the present invention, the exon sequencing primer sequences of the above HLA-B gene are shown in Table 5.

table 5

Preferably, the sequences set forth in SEQ ID NO. 123-136 are used to sequence the full length of the HLA-C gene.

In the present invention, the exon sequencing primer sequences of the above HLA-C gene are shown in Table 6.

Table 6

In a third aspect, the present invention provides a HLA gene sequencing and typing method, the method comprising the following steps:

(1) According to the serotype of the sample, the corresponding group-specific PCR amplification primer according to any one of claims 1 to 5 is selected, and the sample DNA is subjected to PCR amplification and purification to obtain HLA-A and -B. Gene amplification products of and/or -C;

(2) using the full-length sequencing primers of the second aspect, respectively, the purified HLA-A, -B and -C gene amplification products are subjected to sequencing reaction, and purified to obtain a purified sequencing amplification product. ;

(3) The product obtained in the step (2) was electrophoresed in an ABI3730 electrophoresis apparatus to obtain a sequence peak map, which was introduced into the sequence analysis software for analysis in order to perform HLA genotyping.

In a fourth aspect, the invention provides a kit for HLA genotyping comprising: the first aspect The group-specific PCR amplification primers and/or the full-length sequencing primers of the gene as described in the second aspect.

Compared with the prior art, the present invention has the beneficial effects:

In the present application, the HLA gene is subjected to serotyping design group-specific amplification primers, and all the alleles of HLA can be sequenced and classified; the full-length sequence of HLA gene can be amplified and sequenced, and the ambiguity can be effectively solved to achieve an absolute high score; According to the group-specific amplification primer designed by the serologist department, the miss detection can be avoided; the haplotype amplification sequencing results, the single peak is clear at a glance, and the analysis is convenient and quick.

DRAWINGS

1 is a schematic diagram of a strategy for performing amplification using group-specific amplification primers of the HLA-A, -B and -C genes of the present invention and sequencing using the corresponding full-length sequencing primers;

Wherein, the short arrow in the figure indicates a universal sequencing primer; the long arrow in the figure indicates a group-specific amplification primer;

Figure 2 is an electropherogram of the product amplified using MSC301-F\MSC302-R (SEQ ID NO. 65-66);

Figure 3 is a peak image obtained by sequencing using MSC301-F\MSC302-R (SEQ ID NO. 65-66) and sequencing with SEQ ID NO. 123-136.

detailed description

In order to further illustrate the technical means and the effects of the present invention, the technical solutions of the present invention will be further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

The specific techniques or conditions are not indicated in the examples, according to the techniques or conditions described in the literature in the art, or in accordance with the product specifications. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available through formal channels.

A schematic diagram of performing amplification using the group-specific amplification primers of the HLA-A, -B and -C genes of the present invention and sequencing the obtained amplification product using the corresponding full-length sequencing primers is shown in FIG. Arrows indicate universal sequencing primers and long arrows indicate group-specific amplification primers.

Example 1

In this example, an example of HLA-A, -B, and -C absolute high-resolution sequencing typing was performed on 300 patients and their donor specimens requiring hematopoietic stem cell transplantation in Shenzhen.

300 samples of HLA-A, -B, and -C serotypes were selected, and the corresponding group-specific PCR amplification primers and sequencing primers of the present invention were used for sequencing typing to verify the effect of the present invention. .

For each specimen, first, according to its HLA serology, the corresponding HLA-A, -B, and -C full-length sequences of the samples were selected according to the group-specific PCR amplification primers described in the first aspect of the present application. Amplification reaction, amplification reaction conditions: All amplification reactions are set up with the same reaction system, and the composition is as follows:

After setting up the PCR reaction system, all the reactions were simultaneously amplified by the same amplification conditions in the same ABI 9700 type PCR instrument. The cycle parameters were as follows:

After the amplification reaction, all the full-length sequence-specific amplification products of HLA-A, -B, and -C genes were sequenced by 14 positive and reverse walking sequencing primers, and the full length of the gene was determined. The sequencing reaction system is as follows:

The sequencing reaction procedure is:

After the sequencing reaction is completed, electrophoresis is performed in an ABI 3730 sequencer; and the sequencing result is introduced into the sequence analysis software to determine HLA-A, -B, and -C; using this method, a clear judgment of HLA-A, - can be obtained. The absolute high score results of B and -C.

Figure 2 is an electropherogram of the product amplified using MSC301-F\MSC302-R (SEQ ID NO. 65-66), and Figure 3 is a peak image obtained by sequencing using SEQ ID NO. 123-136, which can be seen from Figure 2. The figure shows a specimen with a serological typing of C*01, and the product strip obtained by amplification of MSC301-F\MSC302-R amplification (SEQ ID NO. 65-66) in the present invention is clear, from the figure. 3 It can be seen that after sequencing with SEQ ID NO. 123-136 of the present invention, the single peak is clear, and there is no background peak, and a clear HLA-C absolute high score result can be obtained by introducing the sequence analysis software.

Through the amplification and sequencing primers of the present invention, 300 samples were subjected to sequencing typing experiments, and finally clear HLA-A, -B, and -C absolute high-resolution genotypes (HLA eight-digit nomenclature) were obtained.

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims

A group-specific amplification primer for the HLA gene, wherein the sequence of the PCR amplification primer is selected from the group consisting of the sequences shown in SEQ ID NO. 1-28, the sequence shown in SEQ ID NO. 29-64, and the SEQ. Sequence shown in ID No. 65-94.
The PCR amplification primer according to claim 1, wherein the sequence represented by SEQ ID NO. 1-28 is a primer sequence for amplifying an HLA-A gene;

Preferably, the sequence set forth in SEQ ID NO. 1-2 is for amplifying all alleles of the HLA-A* and A*36 sublines;

Preferably, the sequence set forth in SEQ ID NO. 3-4 is for amplifying all alleles of the HLA-A*02, A*68, A*69 and A*80 sublines;

Preferably, the sequence set forth in SEQ ID NO. 5-6 is for amplifying all alleles of the HLA-A*02 and A*69 sublines;

Preferably, the sequences set forth in SEQ ID NO. 7-8 are for amplifying all alleles of the HLA-A*01, A*36, A*68, A*69 and A*80 sublines;

Preferably, the sequence set forth in SEQ ID NO. 9-10 is for amplifying all alleles of the HLA-A*23 and A*24 sublines;

Preferably, the sequence set forth in SEQ ID NO. 11-12 is for amplifying all alleles of the HLA-A*31 and A*33 sublines;

Preferably, the sequences set forth in SEQ ID NO. 13-14 are all alleles used to amplify the HLA-A*29, A*32 and A*74 sublines;

Preferably, the sequences set forth in SEQ ID NO. 15-16 are all alleles used to amplify the HLA-A*03 and A*30 sublines;

Preferably, the sequences set forth in SEQ ID NO. 17-18 are used to amplify HLA-A*01, A*02, A*03, A*11, A*36, A*68, A*69. And all alleles of the A*80 subfamily;

Preferably, the sequence set forth in SEQ ID NO. 19-20 is for amplifying all alleles of the HLA-A*30 subline;

Preferably, the sequence set forth in SEQ ID NO. 21-22 is for amplifying all alleles of the HLA-A*11 subline;

Preferably, the sequence set forth in SEQ ID NO. 23-24 is for amplifying all alleles of the HLA-A*29 and A*80 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 25-26 are used to amplify HLA-A*25, A*26, A*29, A*31, A*32, A*33, A*34. , all alleles of the A*66, A*74 and A*43 subfamilies;

Preferably, the sequences set forth in SEQ ID NO. 27-28 are alleles used to amplify all serologist lines of HLA-A.
The PCR amplification primer according to claim 1 or 2, wherein the sequence shown in SEQ ID NO. 29-64 is a primer sequence for amplifying an HLA-B gene;

Preferably, the sequences set forth in SEQ ID NO. 29-30 are all alleles used to amplify the HLA-B*07 and B*48 sublines;

Preferably, the sequence set forth in SEQ ID NO. 31-32 is for amplifying all alleles of the HLA-B*08 subline;

Preferably, the sequence set forth in SEQ ID NO. 33-34 is for amplifying all alleles of the HLA-B*13 subline;

Preferably, the sequences set forth in SEQ ID NO. 35-36 are all alleles used to amplify the HLA-B*15 and B*46 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 37-38 are for amplifying all alleles of the HLA-B*14, B*38, B*39 and B*67 sublines;

Preferably, the sequences set forth in SEQ ID NO. 39-40 are used to amplify HLA-B*15, B*51 And all alleles of the B*52 subfamily;

Preferably, the sequences set forth in SEQ ID NOS. 41-42 are all alleles used to amplify the HLA-B*35 and B*73 sublines;

Preferably, the sequence set forth in SEQ ID NOS. 43-44 is for amplifying all alleles of the HLA-B*18, B*44, B*73 and B*82 sublines;

Preferably, the sequence set forth in SEQ ID NO. 45-46 is for amplifying all alleles of the HLA-B*07, B*40:01 and B*81 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 47-48 are for amplifying all alleles of the HLA-B*18, B*27, B*37 and B*40:02/06 sublines;

Preferably, the sequence set forth in SEQ ID NO. 49-50 is for amplifying all alleles of the HLA-B*41, B*45, B*49 and B*50 sublines;

Preferably, the sequence set forth in SEQ ID NO. 51-52 is for amplifying all alleles of the HLA-B*42 subline;

Preferably, the sequence set forth in SEQ ID NO. 53-54 is for amplifying all alleles of the HLA-B*54, B*55, B*56 and B*59 sublines;

Preferably, the sequence set forth in SEQ ID NO. 55-56 is for amplifying all alleles of the HLA-B*57 subline;

Preferably, the sequences set forth in SEQ ID NOS. 57-58 are used to amplify HLA-B*18, B*27, B*35, B*37, B*40, B*41, B*45 , all alleles of the B*49, B*50, B*51, B*52, B*53, B*58 and B*78 subfamilies;

Preferably, the sequences set forth in SEQ ID NO. 59-60 are for amplifying all of the HLA-B*07, B*08, B*15, B*42, B*44 and B*47 sublines. Allele

Preferably, the sequences set forth in SEQ ID NOS. 61-62 are used to amplify HLA-B*41, B*45, All alleles of the B*49, B*50, B*51, B*52, B*53, B*58 and B*78 subfamilies;

Preferably, the sequences set forth in SEQ ID NOS. 63-64 are alleles used to amplify all serologist lines of HLA-B*.
The PCR amplification primer according to any one of claims 1 to 3, wherein the sequence shown in SEQ ID NO. 65-94 is a primer sequence for amplifying an HLA-C gene;

Preferably, the sequences set forth in SEQ ID NO. 65-66 are all alleles used to amplify the HLA-C*01 and C*02 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 67-68 are all alleles used to amplify the HLA-C*01 subline;

Preferably, the sequence set forth in SEQ ID NO. 69-70 is for amplifying all alleles of the HLA-C*02 subline;

Preferably, the sequences set forth in SEQ ID NO. 71-72 are all alleles used to amplify the HLA-C*03 subline;

Preferably, the sequences set forth in SEQ ID NOS. 73-74 are all alleles used to amplify the HLA-C*03 subline;

Preferably, the sequences set forth in SEQ ID NOS. 75-76 are all alleles used to amplify the HLA-C*04 and C*18 sublines;

Preferably, the sequence set forth in SEQ ID NO. 77-78 is for amplifying all alleles of the HLA-C*05 and C*08 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 79-80 are for amplifying all alleles of the HLA-C*06 and C*12 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 81-82 are all alleles used to amplify the HLA-C*07 and C*18 sublines;

Preferably, the sequences set forth in SEQ ID NOS. 83-84 are all alleles used to amplify the HLA-C*07 subline;

Preferably, the sequences set forth in SEQ ID NOS. 85-86 are all alleles used to amplify the HLA-C*14 subline;

Preferably, the sequence set forth in SEQ ID NO. 87-88 is for amplifying all alleles of the HLA-C*15 subline;

Preferably, the sequence set forth in SEQ ID NO. 89-90 is for amplifying all alleles of the HLA-C*16 subline;

Preferably, the sequence set forth in SEQ ID NO. 91-92 is for amplifying all alleles of the HLA-C*17 subline;

Preferably, the sequence set forth in SEQ ID NO. 93-94 is an allele used to amplify all serologist lines of HLA-C.
The PCR amplification primer according to any one of claims 1 to 4, wherein the product fragment obtained by PCR amplification using the PCR amplification primer comprises the corresponding HLA-A, -B and -C All exons, introns, and regulatory regions at both ends of the gene.
A full-length sequencing primer for HLA-A, -B, and -C genes amplified using the PCR amplification primer of any one of claims 1 to 5, characterized in that the full-length sequencing primer of the gene The sequence is selected from the group consisting of the sequences set forth in SEQ ID NO. 95-108, the sequences set forth in SEQ ID NO. 109-122 and the sequences set forth in SEQ ID NO. 123-136.
The full-length sequencing primer for a gene according to claim 6, wherein the sequence of SEQ ID NOS. 95-108 is used for sequencing the full length of the HLA-A gene;

Preferably, the sequence set forth in SEQ ID NO. 109-122 is used to sequence the full length of the HLA-B gene;

Preferably, the sequences set forth in SEQ ID NO. 123-136 are used to sequence the full length of the HLA-C gene.
A HLA gene sequencing typing method, characterized in that the method comprises the following steps:

(1) According to the serotype of the sample, the corresponding group-specific PCR amplification primer according to any one of claims 1 to 5 is selected, and the sample DNA is subjected to PCR amplification and purification to obtain HLA-A and -B. Gene amplification products of and/or -C;

(2) using the full-length sequencing primers of the gene according to claim 6 or 7, respectively, the purified gene amplification products of HLA-A, -B and -C are subjected to sequencing reaction, purified, and purified and sequenced. Add product

(3) The sequencing product obtained in the step (2) was electrophoresed in an ABI3730 electrophoresis apparatus to obtain a sequence peak map, which was introduced into the sequence analysis software for analysis to perform HLA genotyping.
A kit for HLA genotyping, comprising: the group-specific PCR amplification primer according to any one of claims 1 to 5 and/or the full length of the gene according to claim 6 or Sequencing primers.