A genotyping primer set, kit and genotyping method for HLA ultra-high resolution level

Abstract

The present invention relates to the field of biomedical testing technology, and in particular to a primer set, kit, and genotyping method for accurately achieving ultra-high resolution genotyping of 11 human leukocyte antigen (HLA) loci based on nanopore sequencing technology. The present invention provides a primer set for ultra-high resolution HLA genotyping, which is used for multiplex amplification of HLA genes and includes primer pairs for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci. The primer set of the present invention for multiplex amplification of 11 genes for HLA typing can achieve simultaneous amplification of each gene at the HLA gene HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 loci in a single reaction system, thereby improving the integrity, efficiency and uniformity of gene amplification and the accuracy of subsequent HLA genotyping.

Description

Technical Field

[0001] The present invention relates to the field of biomedical testing technology, and in particular to a genotyping primer set, a kit, and a genotyping method for accurately achieving ultra-high resolution of 11 human leukocyte antigen (HLA) sites based on nanopore sequencing technology. Background Art

[0002] The human leukocyte antigen (HLA) system is one of the most complex and highly polymorphic regions in the human genome. It plays a decisive role in antigen recognition, antigen presentation, immune response and regulation, and destruction of foreign antigen target cells. It is also the main material basis for immune rejection reactions. HLA class I and class II antigens on the surface of transplant cells are highly polymorphic and highly immunogenic cell surface markers with significant potential for antigen presentation and immune activation. Therefore, whether the HLA typing between the donor and the recipient is the key to the successful transplantation of allogeneic organs, tissues, or cells. Many studies have shown that with higher resolution of the HLA typing system, the transplant survival rate is higher and the recurrence rate is lower.

[0003] Traditional HLA typing methods, such as serological methods, struggle to accurately distinguish highly similar antigenic subtypes. Molecular detection methods, such as PCR-sequence-specific oligonucleotide probe (PCR-SSOP), PCR-sequence-specific primer (PCR-SSP), and PCR-sequencing typing (PCR-SBT), suffer from numerous drawbacks, including limited resolution, inability to detect novel alleles, cumbersome procedures, and susceptibility to contamination. These drawbacks significantly restrict the demand for accurate HLA typing in both clinical and scientific research scenarios.

[0004] With the advancement of sequencing technology, next-generation sequencing (NGS) has been successfully used for HLA typing. Compared to PCR-based and first-generation sequencing technologies, second-generation sequencing-based HLA typing methods significantly improve the resolution of HLA typing. However, due to the short read length of NGS, ambiguous results can still occur for complex regions of the HLA gene.

[0005] In recent years, nanopore sequencing has emerged as a rapidly developing technology. Using nanoscale protein pores, nucleic acid molecules are passed through them one by one under the influence of an electric field, and the resulting changes in the current signal generated by the bases passing through are monitored in real time to complete the sequencing. This technology offers exceptionally long read lengths, making it particularly well-suited for analyzing complex genomic regions and offering new insights into the challenges of HLA typing. Multiple studies have demonstrated that nanopore sequencing-based HLA typing methods can sequence the entire length of multiple HLA loci, significantly improving the resolution of some HLA loci. However, current studies have limited the full-length amplification of the 5'UTR to 3'UTR for HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPB1, and HLA-DPA1 loci. For HLA-DRB1 and -DRB3 / 4 / 5 loci, primer design is challenging due to the high similarity of some regions and the varying number and relative arrangement of individual monomeric genes. Currently, primer design is typically performed in segments or by multiple amplification tubes. HLA is the most complex human genetic polymorphism system known to date. The HLA genetic region contains not only numerous genetic loci, but also a large number of alleles at each locus. China has a large population base, numerous ethnic groups, and a wide geographical distribution, resulting in a large number of variations. However, existing HLA analysis-related websites and software generally contain relatively low proportions of data on the Chinese population. Therefore, traditional primer design methods suffer from poor inclusiveness, leading to amplification failures, biases, and missed detections in some special population groups. There are also several long-PCR enzymes currently on the market, but these enzymes have low overall amplification efficiency, with one round of amplification taking approximately 8 hours. These combined factors bring numerous inconveniences to experiments, increase the risk of contamination, and increase experimental time and price costs. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of the prior art by providing a primer set and genotyping method for ultra-high-resolution HLA genotyping. The primer set of the present invention, which is used for multiplex amplification of 11 HLA genes, enables simultaneous amplification of the HLA genes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci in a single reaction system, thereby improving the integrity, efficiency, and uniformity of gene amplification, as well as the accuracy of subsequent HLA genotyping.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a primer set for ultra-high-resolution HLA genotyping is provided. The primer set is used for multiplex amplification of HLA genes; the primer set includes primer pairs for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci. To improve primer inclusiveness, Scheme 1 employs an Ω primer design and a nested primer design. This scheme covers the full-length exon sequences of 11 loci. The upstream and downstream primers of the primer pair and the associated nucleotide sequences are selected from any one or more of the following groups 1 to 48:

[0009] The upstream and downstream primer sequences of the primer pairs for each site in Group 1 are as follows:

[0010] 1) The HLA-AF sequence is shown in SEQ ID NO. 1, the HLA-A-R1 sequence is shown in SEQ ID NO. 2, and the HLA-A-R2 sequence is shown in SEQ ID NO. 3;

[0011] 2) The HLA-BF sequence is shown in SEQ ID NO. 4, the HLA-B-R1 sequence is shown in SEQ ID NO. 5, and the HLA-B-R2 sequence is shown in SEQ ID NO. 6;

[0012] 3) The HLA-CF sequence is shown in SEQ ID NO. 7, the HLA-C-R1 sequence is shown in SEQ ID NO. 8, and the HLA-C-R2 sequence is shown in SEQ ID NO. 9;

[0013] 4) The HLA-DPA-F1 sequence is shown in SEQ ID NO. 10, the HLA-DPA-F2 sequence is shown in SEQ ID NO. 11, and the HLA-DPA-R sequence is shown in SEQ ID NO. 12;

[0014] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 13, the HLA-DPB-R1 sequence is shown in SEQ ID NO. 14, and the HLA-DPB-R2 sequence is shown in SEQ ID NO. 15;

[0015] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 16, the HLA-DQA-R1 sequence is shown in SEQ ID NO. 17, and the HLA-DQA-R2 sequence is shown in SEQ ID NO. 18;

[0016] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 19, the HLA-DQB-R1 sequence is shown in SEQ ID NO. 20, and the HLA-DQB-R2 sequence is shown in SEQ ID NO. 21;

[0017] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 22, the HLA-DRB1345-R1 sequence is shown in SEQ ID NO. 23, and the HLA-DRB1345-R2 sequence is shown in SEQ ID NO. 24;

[0018] The upstream and downstream primer sequences of the primer pairs for each site in Group 2 are as follows:

[0019] 1) The HLA-AF sequence is shown in SEQ ID NO. 25, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0020] 2) The HLA-BF sequence is shown in SEQ ID NO. 26, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0021] 3) The HLA-CF sequence is shown in SEQ ID NO. 27, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0022] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 28, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0023] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 29, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0024] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 30, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0025] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 31, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0026] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 32, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0027] The upstream and downstream primer sequences of the primer pairs for each site in Group 3 are as follows:

[0028] 1) The HLA-AF sequence is shown in SEQ ID NO. 33, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0029] 2) The HLA-BF sequence is shown in SEQ ID NO. 34, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0030] 3) The HLA-CF sequence is shown in SEQ ID NO. 35, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0031] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 36, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0032] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 37, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0033] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 38, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0034] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 39, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0035] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 40, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0036] The upstream and downstream primer sequences of the primer pairs for each site in Group 4 are as follows:

[0037] 1) The HLA-AF sequence is shown in SEQ ID NO. 41, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0038] 2) The HLA-BF sequence is shown in SEQ ID NO. 42, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0039] 3) The HLA-CF sequence is shown in SEQ ID NO. 43, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0040] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 44, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0041] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 45, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0042] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 46, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0043] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 47, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0044] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 48, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0045] The upstream and downstream primer sequences of the primer pairs for each site in Group 5 are as follows:

[0046] 1) The HLA-AF sequence is shown in SEQ ID NO. 49, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0047] 2) The HLA-BF sequence is shown in SEQ ID NO. 50, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0048] 3) The HLA-CF sequence is shown in SEQ ID NO. 51, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0049] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 52, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0050] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 53, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0051] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 54, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0052] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 55, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0053] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 56, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0054] The upstream and downstream primer sequences of the primer pairs for each site in Group 6 are as follows:

[0055] 1) The HLA-AF sequence is shown in SEQ ID NO. 57, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0056] 2) The HLA-BF sequence is shown in SEQ ID NO. 58, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0057] 3) The HLA-CF sequence is shown in SEQ ID NO. 59, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0058] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 60, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0059] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 61, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0060] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 62, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0061] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 63, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0062] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 64, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0063] The upstream and downstream primer sequences of the primer pairs for each site in Group 7 are as follows:

[0064] 1) The HLA-AF sequence is shown in SEQ ID NO. 65, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0065] 2) The HLA-BF sequence is shown in SEQ ID NO. 66, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0066] 3) The HLA-CF sequence is shown in SEQ ID NO. 67, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0067] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 68, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0068] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 69, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0069] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 70, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0070] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 71, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0071] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 72, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0072] The upstream and downstream primer sequences of the primer pairs for each site in Group 8 are as follows:

[0073] 1) The HLA-AF sequence is shown in SEQ ID NO. 73, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0074] 2) The HLA-BF sequence is shown in SEQ ID NO. 74, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0075] 3) The HLA-CF sequence is shown in SEQ ID NO. 75, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0076] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 76, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0077] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 77, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0078] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 78, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0079] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 79, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0080] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 80, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0081] The upstream and downstream primer sequences of the primer pairs for each site in Group 9 are as follows:

[0082] 1) The HLA-AF sequence is shown in SEQ ID NO. 81, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0083] 2) The HLA-BF sequence is shown in SEQ ID NO. 82, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0084] 3) The HLA-CF sequence is shown in SEQ ID NO. 83, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0085] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 84, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0086] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 85, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0087] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 86, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0088] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 87, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0089] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 88, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0090] The upstream and downstream primer sequences of the primer pairs for each site in group 10 are as follows:

[0091] 1) The HLA-AF sequence is shown in SEQ ID NO. 89, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0092] 2) The HLA-BF sequence is shown in SEQ ID NO. 90, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0093] 3) The HLA-CF sequence is shown in SEQ ID NO. 91, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0094] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 92, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0095] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 93, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0096] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 94, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0097] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 95, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0098] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 96, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0099] The upstream and downstream primer sequences of the primer pairs for each site in group 11 are as follows:

[0100] 1) The HLA-AF sequence is shown in SEQ ID NO. 97, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0101] 2) The HLA-BF sequence is shown in SEQ ID NO. 98, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0102] 3) The HLA-CF sequence is shown in SEQ ID NO. 99, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0103] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 100, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0104] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 101, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0105] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 102, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0106] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 103, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0107] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 104, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0108] The upstream and downstream primer sequences of the primer pairs for each site in Group 12 are as follows:

[0109] 1) The HLA-AF sequence is shown in SEQ ID NO. 105, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0110] 2) The HLA-BF sequence is shown in SEQ ID NO. 106, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0111] 3) The HLA-CF sequence is shown in SEQ ID NO. 107, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0112] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 108, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0113] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 109, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0114] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 110, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0115] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 111, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0116] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 112, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0117] The upstream and downstream primer sequences of the primer pairs for each site in Group 13 are as follows:

[0118] 1) The HLA-AF sequence is shown in SEQ ID NO. 113, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0119] 2) The HLA-BF sequence is shown in SEQ ID NO. 114, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0120] 3) The HLA-CF sequence is shown in SEQ ID NO. 115, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0121] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 116, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0122] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 117, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0123] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 118, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0124] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 119, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0125] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 120, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0126] The upstream and downstream primer sequences of the primer pairs for each site in Group 14 are as follows:

[0127] 1) The HLA-AF sequence is shown in SEQ ID NO. 121, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0128] 2) The HLA-BF sequence is shown in SEQ ID NO. 122, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0129] 3) The HLA-CF sequence is shown in SEQ ID NO. 123, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0130] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 124, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0131] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 125, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0132] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 126, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0133] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 127, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0134] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 128, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0135] The upstream and downstream primer sequences of the primer pairs for each site in Group 15 are as follows:

[0136] 1) The HLA-AF sequence is shown in SEQ ID NO. 129, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0137] 2) The HLA-BF sequence is shown in SEQ ID NO. 130, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0138] 3) The HLA-CF sequence is shown in SEQ ID NO. 131, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0139] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 132, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0140] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 133, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0141] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 134, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0142] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 135, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0143] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 136, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0144] The upstream and downstream primer sequences of the primer pairs for each site in Group 16 are as follows:

[0145] 1) The HLA-AF sequence is shown in SEQ ID NO. 137, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0146] 2) The HLA-BF sequence is shown in SEQ ID NO. 138, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0147] 3) The HLA-CF sequence is shown in SEQ ID NO. 139, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0148] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 140, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0149] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 141, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0150] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 142, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0151] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 143, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0152] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 144, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0153] The upstream and downstream primer sequences of the primer pairs for each site in Group 17 are as follows:

[0154] 1) The HLA-AF sequence is shown in SEQ ID NO. 145, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0155] 2) The HLA-BF sequence is shown in SEQ ID NO. 146, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0156] 3) The HLA-CF sequence is shown in SEQ ID NO. 147, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0157] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 148, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0158] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 149, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0159] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 150, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0160] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 151, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0161] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 152, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0162] The upstream and downstream primer sequences of the primer pairs for each site in Group 18 are as follows:

[0163] 1) The HLA-AF sequence is shown in SEQ ID NO. 153, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0164] 2) The HLA-BF sequence is shown in SEQ ID NO. 154, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0165] 3) The HLA-CF sequence is shown in SEQ ID NO. 155, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0166] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 156, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0167] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 157, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0168] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 158, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0169] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 159, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0170] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 160, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0171] The upstream and downstream primer sequences of the primer pairs for each site in Group 19 are as follows:

[0172] 1) The HLA-AF sequence is shown in SEQ ID NO. 161, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0173] 2) The HLA-BF sequence is shown in SEQ ID NO. 162, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0174] 3) The HLA-CF sequence is shown in SEQ ID NO. 163, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0175] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 164, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0176] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 165, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0177] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 166, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0178] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 167, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0179] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 168, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0180] The upstream and downstream primer sequences of the primer pairs for each site in Group 20 are as follows:

[0181] 1) The HLA-AF sequence is shown in SEQ ID NO. 169, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0182] 2) The HLA-BF sequence is shown in SEQ ID NO. 170, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0183] 3) The HLA-CF sequence is shown in SEQ ID NO. 171, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0184] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 172, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0185] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 173, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0186] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 174, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0187] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 175, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0188] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 176, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0189] The upstream and downstream primer sequences of the primer pairs for each site in Group 21 are as follows:

[0190] 1) The HLA-AF sequence is shown in SEQ ID NO. 177, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0191] 2) The HLA-BF sequence is shown in SEQ ID NO. 178, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0192] 3) The HLA-CF sequence is shown in SEQ ID NO. 179, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0193] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 180, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0194] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 181, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0195] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 182, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0196] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 183, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0197] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 184, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0198] The upstream and downstream primer sequences of the primer pairs for each site in Group 22 are as follows:

[0199] 1) The HLA-AF sequence is shown in SEQ ID NO. 185, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0200] 2) The HLA-BF sequence is shown in SEQ ID NO. 186, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0201] 3) The HLA-CF sequence is shown in SEQ ID NO. 187, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0202] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 188, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0203] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 189, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0204] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 190, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0205] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 191, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0206] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 192, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0207] The upstream and downstream primer sequences of the primer pairs for each site in Group 23 are as follows:

[0208] 1) The HLA-AF sequence is shown in SEQ ID NO. 193, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0209] 2) The HLA-BF sequence is shown in SEQ ID NO. 194, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0210] 3) The HLA-CF sequence is shown in SEQ ID NO. 195, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0211] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 196, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0212] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 197, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0213] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 198, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0214] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 199, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0215] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 200, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0216] The upstream and downstream primer sequences of the primer pairs for each site in Group 24 are as follows:

[0217] 1) The HLA-AF sequence is shown in SEQ ID NO. 201, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0218] 2) The HLA-BF sequence is shown in SEQ ID NO. 202, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0219] 3) The HLA-CF sequence is shown in SEQ ID NO. 203, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0220] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 204, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0221] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 205, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0222] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 206, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0223] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 207, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0224] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 208, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0225] The upstream and downstream primer sequences of the primer pairs for each site in group 25 are as follows:

[0226] 1) The HLA-AF sequence is shown in SEQ ID NO. 209, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0227] 2) The HLA-BF sequence is shown in SEQ ID NO. 210, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0228] 3) The HLA-CF sequence is shown in SEQ ID NO. 211, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0229] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 212, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0230] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 213, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0231] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 214, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0232] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 215, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0233] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 216, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0234] The upstream and downstream primer sequences of the primer pairs for each site in Group 26 are as follows:

[0235] 1) The HLA-AF sequence is shown in SEQ ID NO. 217, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0236] 2) The HLA-BF sequence is shown in SEQ ID NO. 218, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0237] 3) The HLA-CF sequence is shown in SEQ ID NO. 219, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0238] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 220, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0239] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 221, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0240] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 222, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0241] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 223, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0242] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 224, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0243] The upstream and downstream primer sequences of the primer pairs for each site in Group 27 are as follows:

[0244] 1) The HLA-AF sequence is shown in SEQ ID NO. 225, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0245] 2) The HLA-BF sequence is shown in SEQ ID NO. 226, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0246] 3) The HLA-CF sequence is shown in SEQ ID NO. 227, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0247] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 228, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0248] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 229, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0249] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 230, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0250] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 231, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0251] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 232, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0252] The upstream and downstream primer sequences of the primer pairs for each site in Group 28 are as follows:

[0253] 1) The HLA-AF sequence is shown in SEQ ID NO. 233, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0254] 2) The HLA-BF sequence is shown in SEQ ID NO. 234, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0255] 3) The HLA-CF sequence is shown in SEQ ID NO. 235, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0256] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 236, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0257] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 237, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0258] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 238, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0259] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 239, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0260] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 240, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0261] The upstream and downstream primer sequences of the primer pairs for each site in Group 29 are as follows:

[0262] 1) The HLA-AF sequence is shown in SEQ ID NO. 241, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0263] 2) The HLA-BF sequence is shown in SEQ ID NO. 242, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0264] 3) The HLA-CF sequence is shown in SEQ ID NO. 243, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0265] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 244, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0266] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 245, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0267] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 246, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0268] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 247, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0269] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 248, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0270] The upstream and downstream primer sequences of the primer pairs for each site in group 30 are as follows:

[0271] 1) The HLA-AF sequence is shown in SEQ ID NO. 249, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0272] 2) The HLA-BF sequence is shown in SEQ ID NO. 250, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0273] 3) The HLA-CF sequence is shown in SEQ ID NO. 251, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0274] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 252, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0275] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 253, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0276] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 254, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0277] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 255, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0278] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 256, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0279] The upstream and downstream primer sequences of the primer pairs for each site in group 31 are as follows:

[0280] 1) The HLA-AF sequence is shown in SEQ ID NO. 257, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0281] 2) The HLA-BF sequence is shown in SEQ ID NO. 258, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0282] 3) The HLA-CF sequence is shown in SEQ ID NO. 259, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0283] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 260, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0284] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 261, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0285] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 262, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0286] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 263, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0287] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 264, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0288] The upstream and downstream primer sequences of the primer pairs for each site in group 32 are as follows:

[0289] 1) The HLA-AF sequence is shown in SEQ ID NO. 265, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0290] 2) The HLA-BF sequence is shown in SEQ ID NO. 266, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0291] 3) The HLA-CF sequence is shown in SEQ ID NO. 267, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0292] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 268, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0293] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 269, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0294] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 270, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0295] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 271, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0296] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 272, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0297] The upstream and downstream primer sequences of the primer pairs for each site in group 33 are as follows:

[0298] 1) The HLA-AF sequence is shown in SEQ ID NO. 273, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0299] 2) The HLA-BF sequence is shown in SEQ ID NO. 274, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0300] 3) The HLA-CF sequence is shown in SEQ ID NO. 275, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0301] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 276, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0302] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 277, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0303] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 278, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0304] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 279, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0305] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 280, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0306] The upstream and downstream primer sequences of the primer pairs for each site in group 34 are as follows:

[0307] 1) The HLA-AF sequence is shown in SEQ ID NO. 281, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0308] 2) The HLA-BF sequence is shown in SEQ ID NO. 282, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0309] 3) The HLA-CF sequence is shown in SEQ ID NO. 283, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0310] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 284, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0311] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 285, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0312] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 286, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0313] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 287, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0314] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 288, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0315] The upstream and downstream primer sequences of the primer pairs for each site in group 35 are as follows:

[0316] 1) The HLA-AF sequence is shown in SEQ ID NO. 289, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0317] 2) The HLA-BF sequence is shown in SEQ ID NO. 290, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0318] 3) The HLA-CF sequence is shown in SEQ ID NO. 291, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0319] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 292, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0320] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 293, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0321] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 294, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0322] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 295, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0323] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 296, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0324] The upstream and downstream primer sequences of the primer pairs for each site in group 36 are as follows:

[0325] 1) The HLA-AF sequence is shown in SEQ ID NO. 297, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0326] 2) The HLA-BF sequence is shown in SEQ ID NO. 298, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0327] 3) The HLA-CF sequence is shown in SEQ ID NO. 299, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0328] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 300, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0329] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 301, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0330] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 302, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0331] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 303, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0332] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 304, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0333] The upstream and downstream primer sequences of the primer pairs for each site in group 37 are as follows:

[0334] 1) The HLA-AF sequence is shown in SEQ ID NO. 305, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0335] 2) The HLA-BF sequence is shown in SEQ ID NO. 306, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0336] 3) The HLA-CF sequence is shown in SEQ ID NO. 307, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0337] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 308, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0338] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 309, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0339] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 310, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0340] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 311, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0341] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 312, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0342] The upstream and downstream primer sequences of the primer pairs for each site in group 38 are as follows:

[0343] 1) The HLA-AF sequence is shown in SEQ ID NO. 313, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0344] 2) The HLA-BF sequence is shown in SEQ ID NO. 314, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0345] 3) The HLA-CF sequence is shown in SEQ ID NO. 315, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0346] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 316, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0347] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 317, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0348] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 318, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0349] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 319, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0350] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 320, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0351] The upstream and downstream primer sequences of the primer pairs for each site in group 39 are as follows:

[0352] 1) The HLA-AF sequence is shown in SEQ ID NO. 321, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0353] 2) The HLA-BF sequence is shown in SEQ ID NO. 322, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0354] 3) The HLA-CF sequence is shown in SEQ ID NO. 323, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0355] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 324, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0356] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 325, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0357] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 326, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0358] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 327, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0359] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 328, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0360] The upstream and downstream primer sequences of the primer pairs for each site in group 40 are as follows:

[0361] 1) The HLA-AF sequence is shown in SEQ ID NO. 329, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0362] 2) The HLA-BF sequence is shown in SEQ ID NO. 330, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0363] 3) The HLA-CF sequence is shown in SEQ ID NO. 331, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0364] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 332, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0365] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 333, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0366] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 334, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0367] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 335, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0368] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 336, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0369] The upstream and downstream primer sequences of the primer pairs for each site in group 41 are as follows:

[0370] 1) The HLA-AF sequence is shown in SEQ ID NO. 337, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0371] 2) The HLA-BF sequence is shown in SEQ ID NO. 338, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0372] 3) The HLA-CF sequence is shown in SEQ ID NO. 339, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0373] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 340, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0374] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 341, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0375] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 342, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0376] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 343, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0377] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 344, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0378] The upstream and downstream primer sequences of the primer pairs for each site in group 42 are as follows:

[0379] 1) The HLA-AF sequence is shown in SEQ ID NO. 345, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0380] 2) The HLA-BF sequence is shown in SEQ ID NO. 346, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0381] 3) The HLA-CF sequence is shown in SEQ ID NO. 347, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0382] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 348, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0383] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 349, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0384] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 350, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0385] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 351, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0386] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 352, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0387] The upstream and downstream primer sequences of the primer pairs for each site in group 43 are as follows:

[0388] 1) The HLA-AF sequence is shown in SEQ ID NO. 353, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0389] 2) The HLA-BF sequence is shown in SEQ ID NO. 354, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0390] 3) The HLA-CF sequence is shown in SEQ ID NO. 355, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0391] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 356, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0392] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 357, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0393] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 358, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0394] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 359, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0395] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 360, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0396] The upstream and downstream primer sequences of the primer pairs for each site in group 44 are as follows:

[0397] 1) The HLA-AF sequence is shown in SEQ ID NO. 361, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0398] 2) The HLA-BF sequence is shown in SEQ ID NO. 362, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0399] 3) The HLA-CF sequence is shown in SEQ ID NO. 363, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0400] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 364, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0401] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 365, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0402] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 366, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0403] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 367, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0404] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 368, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0405] The upstream and downstream primer sequences of the primer pairs for each site in group 45 are as follows:

[0406] 1) The HLA-AF sequence is shown in SEQ ID NO. 369, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0407] 2) The HLA-BF sequence is shown in SEQ ID NO. 370, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0408] 3) The HLA-CF sequence is shown in SEQ ID NO. 371, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0409] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 372, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0410] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 373, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0411] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 374, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0412] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 375, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0413] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 376, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0414] The upstream and downstream primer sequences of the primer pairs for each site in group 46 are as follows:

[0415] 1) The HLA-AF sequence is shown in SEQ ID NO. 377, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0416] 2) The HLA-BF sequence is shown in SEQ ID NO. 378, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0417] 3) The HLA-CF sequence is shown in SEQ ID NO. 379, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0418] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 380, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0419] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 381, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0420] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 382, ​​and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0421] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 383, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0422] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 384, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0423] The upstream and downstream primer sequences of the primer pairs for each site in group 47 are as follows:

[0424] 1) The HLA-AF sequence is shown in SEQ ID NO. 385, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0425] 2) The HLA-BF sequence is shown in SEQ ID NO. 386, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0426] 3) The HLA-CF sequence is shown in SEQ ID NO. 387, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0427] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 388, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0428] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 389, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0429] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 390, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0430] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 391, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0431] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 392, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0432] The upstream and downstream primer sequences of the primer pairs for each site in group 48 are as follows:

[0433] 1) The HLA-AF sequence is shown in SEQ ID NO. 393, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0434] 2) The HLA-BF sequence is shown in SEQ ID NO. 394, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0435] 3) The HLA-CF sequence is shown in SEQ ID NO. 395, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0436] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 396, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0437] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 397, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0438] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 398, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0439] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 399, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0440] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 400, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in Group 1.

[0441] In a second aspect, the present invention provides another primer set for ultra-high-resolution HLA genotyping, also used for multiplex HLA gene amplification. The primer set includes primer pairs for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci. To improve primer inclusiveness, Scheme 2 employs a nested primer design, which covers the full-length exon sequences of 11 loci. The upstream and downstream primers and their associated nucleotide sequences are shown in the table below.

[0442] The site can be set up into two sets of upstream and downstream primers. The upstream and downstream primers and their related nucleotide sequences are shown in the following table:

[0443]

[0444]

[0445] In a third aspect of the present invention, there is provided use of the primer set described in the first aspect in preparing an HLA ultra-high resolution genotyping detection kit.

[0446] In a fourth aspect of the present invention, an HLA ultra-high resolution genotyping detection kit is provided, wherein the detection kit comprises the primer set and detection reagent as described in the first aspect.

[0447] Preferably, the detection reagent includes the HLA gene rapid amplification enzyme system Flash PCR Master MixFor Long Fragment.

[0448] In a fifth aspect of the present invention, there is provided use of the primer set described in the second aspect in preparing an HLA ultra-high resolution genotyping detection kit.

[0449] In a sixth aspect of the present invention, an HLA ultra-high resolution genotyping detection kit is provided, wherein the detection kit comprises the primer set and detection reagent as described in the second aspect.

[0450] Preferably, the detection reagent includes the HLA gene rapid amplification enzyme system Flash PCR Master MixFor Long Fragment and Index synthesis related oligonucleotide sequences;

[0451] The Index synthesis-related oligonucleotide sequences are selected from any one or more groups of Index01-Index48. The upstream and downstream primers of the primer pairs of Index01-Index48 and the related nucleotide sequences are shown in the following table:

[0452]

[0453]

[0454]

[0455]

[0456] In a seventh aspect of the present invention, a full-length and rapid sequencing method for HLA typing based on nanopore sequencing is provided, which comprises using the above-mentioned primer set or kit to amplify the HLA gene in the sample to be tested, and then sequencing the amplified product.

[0457] In an eighth aspect of the present invention, a method for HLA genotyping for non-diagnostic and non-therapeutic purposes is provided. The method comprises using the above-mentioned primer set or kit to amplify the HLA genes in a test sample, constructing a sequencing library from the amplified products, and then sequencing and performing bioinformatics analysis on the sequencing data to obtain full-length, ultra-high-resolution typing information for all 11 HLA genes in the test sample.

[0458] Compared with the prior art, the present invention has the following beneficial effects:

[0459] The primer set for HLA gene multiplex amplification provided by the present invention performs full-length amplification and full-length direct sequencing of 11 gene loci, including HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1, thereby improving the efficiency of gene amplification and ensuring the integrity and uniformity of sequencing. The full-length direct amplification and direct sequencing of genes directly obtain the allele type of each gene, eliminating the splicing process between different exons, thereby avoiding a large number of ambiguous results generated during first-generation sequencing, second-generation sequencing and analysis, and thereby improving the accuracy of subsequent HLA haplotype genotyping.

[0460] The HLA genotyping method provided by the present invention utilizes the primer set of the first aspect and the rapid amplification enzyme described in the second aspect to rapidly amplify 11 target genes in clinical samples. A rapid library construction method is then used to construct a library of the amplified products, and the library is sequenced using nanopore sequencing technology. The resulting reads provide more complete coverage of the full length of the 11 genes, with more balanced read counts across different genes, offering advantages. Reliable full-length amplification and nanopore sequencing provide strong support for organ transplantation, bone marrow typing, and other applications.

[0461] Specifically, compared with previous HLA detection methods, the technical advantages of the present invention are as follows:

[0462] 1) The present invention provides a primer design method based on omega primers, nested primers, and degenerate bases. The primers or primer combinations designed by this method can maximize the population coverage of the test method, improving the effectiveness of indiscriminate HLA screening. Based on the above method, the present invention designed a primer combination for multiplex amplification of 11 genes for HLA typing. In Scheme 1, i.e., part of Example 1, the primers can be Ω primers, comprising a 3p arm, a 5p arm, and a loop separating the two arms. The 3p arm serves as a promoter for the polymerase extension reaction, the 5p arm serves as an anchor for binding to the DNA template sequence, and the loop separating the two arms contains anchor sequence 1, anchor sequence 2, and a barcode sequence for library splitting. During amplification, both the 3p arm and the 5p arm must specifically bind to the template simultaneously. Mismatches between the 3p arm and the template destabilize the loop structure, causing the 3p arm to detach from the template, reducing the probability of polymerase extension of the primer. Compared to traditional primers, this technology significantly increases primer specificity. The present invention designs a longer 5p arm and includes some degenerate bases, allowing for effective binding even in the presence of mismatches in the template. This feature effectively improves primer tolerance and reduces the risk of PCR failure, missed detection, and allele bias. The nested primers of Option 2, Example 2, also serve the same purpose. While ensuring full-length gene amplification, the nested primer design and partial degenerate bases significantly increase the probability of detecting large populations without causing uneven intergenic read output and allele bias due to the presence of low-frequency SNPs. This primer set can achieve simultaneous amplification of HLA gene HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1 sites in a single reaction system, thereby improving the simplicity of the experiment, as well as the integrity, efficiency and uniformity of gene amplification and the accuracy of subsequent HLA genotyping.

[0463] 2) The present invention adopts Comwe's multiple-blocked long-fragment PCR amplification enzyme system, Flash PCR MasterMix For Long Fragment (Comwe's commercial product, item number CW3397), which contains Super DNA Polymerase, dNTP, unique extension factors and a high-performance optimized buffer system. Only primers and templates need to be added to perform PCR experiments, reducing the complicated sample addition process. Super DNA Polymerase has both 5′→3′ DNA polymerase activity and 3′→5′ exonuclease activity, and its fidelity is about 80 times that of Taq DNA polymerase, and the amplified product has blunt ends. This enzyme is a high-fidelity enzyme that has undergone multiple blocking. It effectively blocks polymerase activity at 55°C and below, and quickly recovers polymerase activity after incubation at 98°C for 30s. It is suitable for amplifying target fragments of 100bp to 40,000bp, and is particularly suitable for amplifying fragments ≥10,000bp. The amplification speed is as high as 10kb / s, which greatly saves PCR reaction time. The enzyme can complete the amplification of long fragments in a very short time, which can greatly shorten the detection time. It can complete 30 cycles of amplification of DNA fragments of 10,000 to 160,000 bp in about 40 minutes.

[0464] 3) Example 1 of the present invention uses a rapid library construction kit. Omega primers have their own barcodes, so the overall library construction process includes: long-range PCR, PCR product magnetic bead purification and quantification, PCR product mixing, end repair + A, sequencing adapter ligation and magnetic bead purification (see Figure 1 ). Common third-generation library construction methods: long-fragment PCR, PCR product magnetic bead purification and quantification, end repair + A, barcode ligation, library magnetic bead purification and quantification, library mixing, sequencing adapter ligation, magnetic bead purification and quantification (see Figure 1 Compared to the commonly used third-generation library construction method, the rapid method reduces the steps of barcode ligation, library magnetic bead purification, and quantification. Furthermore, after the first round of long-range PCR, samples are directly mixed as a single pooled sample for library construction, significantly reducing the workload of subsequent end-repair + A, library magnetic bead purification, and quantification. While the overall library construction process in Example 2 is more complex than in Example 1, the nested primer design effectively mitigates interference from low-frequency SNPs during HLA population testing, thus laying the foundation for large-scale ultra-high-resolution analysis of potential HLA donors.

[0465] In summary, Examples 1 and 2 of this patent achieve the following advantages over previous HLA detection methods: ① Full-length direct detection of single-gene haplotypes; ② Ultra-fast amplification, library construction, and sequencing; ③ Large population detection, overcoming the interference of low-frequency SNPs; ④ Ultra-high-resolution typing of 11 loci of Class I and II. BRIEF DESCRIPTION OF THE DRAWINGS

[0466] Figure 1 Schematic diagram of the database construction process.

[0467] Figure 2 These are the HLA primer amplification results of three clinical blood samples in Example 1.

[0468] Figure 3 This is the quality inspection result of the linker annealing electrophoresis in Example 2.

[0469] Figure 4 These are the HLA primer amplification results of three clinical blood samples in Example 2. DETAILED DESCRIPTION

[0470] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. However, the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0471] The primers in the examples of the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The remaining components in the kit were commercially available from Jiangsu Kangwei Century Biotechnology Co., Ltd. and can be replaced by other similar commercial products. The genomic DNA extraction in the following examples was performed using the following method:

[0472] Blood samples need to be extracted for gDNA using the Cowin Blood Genomic DNA Mini Kit (0.1-1 mL, cw20877). If automated extraction is used, use the CWY129S-96 reagent. If the sample is DNA, 0.5x Cowin Century DNA Purification Magnetic Beads (Cat. No. CW3171M) need to be used for purification.

[0473] Example 1

[0474] 1. Primer and barcode synthesis

[0475] Primer Design and Synthesis HLA-related gene sequences were searched on NCBI (National Center of Biotechnology Information). Based on the primer design principles for multiplex PCR, specific primer sequences and barcode sequences were designed as shown in Tables 1 and 2 below.

[0476] Table 1.48 barcode sequences

[0477]

[0478]

[0479] Table 2 Primer information

[0480]

[0481]

[0482]

[0483] The present invention provides 48 barcode sequences that can be used to distinguish 48 different samples during sequencing.

[0484] The present invention provides 48 primer sets. The primer pair sequences for each site in each set differ from the primer pair sequences for each site in the first set (see Table 2) only in that the barcode sequence in the Ω primer for each site is different. The Ω primers for each site in the second set contain the barcode_2 sequence, the Ω primers for each site in the third set contain the barcode_3 sequence, and so on.

[0485] The upstream and downstream primer sequences of the primer pairs for each site in Group 2 to Group 48 are as follows:

[0486] The upstream and downstream primer sequences of the primer pairs for each site in Group 2 are as follows:

[0487] 1) The HLA-AF sequence is shown in SEQ ID NO. 25, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0488] 2) The HLA-BF sequence is shown in SEQ ID NO. 26, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0489] 3) The HLA-CF sequence is shown in SEQ ID NO. 27, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0490] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 28, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0491] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 29, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0492] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 30, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0493] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 31, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0494] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 32, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0495] The upstream and downstream primer sequences of the primer pairs for each site in Group 3 are as follows:

[0496] 1) The HLA-AF sequence is shown in SEQ ID NO. 33, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0497] 2) The HLA-BF sequence is shown in SEQ ID NO. 34, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0498] 3) The HLA-CF sequence is shown in SEQ ID NO. 35, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0499] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 36, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0500] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 37, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0501] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 38, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0502] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 39, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0503] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 40, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0504] The upstream and downstream primer sequences of the primer pairs for each site in Group 4 are as follows:

[0505] 1) The HLA-AF sequence is shown in SEQ ID NO. 41, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0506] 2) The HLA-BF sequence is shown in SEQ ID NO. 42, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0507] 3) The HLA-CF sequence is shown in SEQ ID NO. 43, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0508] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 44, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0509] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 45, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0510] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 46, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0511] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 47, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0512] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 48, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0513] The upstream and downstream primer sequences of the primer pairs for each site in Group 5 are as follows:

[0514] 1) The HLA-AF sequence is shown in SEQ ID NO. 49, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0515] 2) The HLA-BF sequence is shown in SEQ ID NO. 50, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0516] 3) The HLA-CF sequence is shown in SEQ ID NO. 51, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0517] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 52, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0518] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 53, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0519] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 54, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0520] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 55, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0521] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 56, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0522] The upstream and downstream primer sequences of the primer pairs for each site in Group 6 are as follows:

[0523] 1) The HLA-AF sequence is shown in SEQ ID NO. 57, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0524] 2) The HLA-BF sequence is shown in SEQ ID NO. 58, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0525] 3) The HLA-CF sequence is shown in SEQ ID NO. 59, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0526] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 60, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0527] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 61, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0528] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 62, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0529] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 63, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0530] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 64, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0531] The upstream and downstream primer sequences of the primer pairs for each site in Group 7 are as follows:

[0532] 1) The HLA-AF sequence is shown in SEQ ID NO. 65, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0533] 2) The HLA-BF sequence is shown in SEQ ID NO. 66, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0534] 3) The HLA-CF sequence is shown in SEQ ID NO. 67, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0535] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 68, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0536] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 69, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0537] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 70, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0538] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 71, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0539] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 72, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0540] The upstream and downstream primer sequences of the primer pairs for each site in Group 8 are as follows:

[0541] 1) The HLA-AF sequence is shown in SEQ ID NO. 73, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0542] 2) The HLA-BF sequence is shown in SEQ ID NO. 74, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0543] 3) The HLA-CF sequence is shown in SEQ ID NO. 75, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0544] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 76, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0545] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 77, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0546] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 78, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0547] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 79, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0548] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 80, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0549] The upstream and downstream primer sequences of the primer pairs for each site in Group 9 are as follows:

[0550] 1) The HLA-AF sequence is shown in SEQ ID NO. 81, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0551] 2) The HLA-BF sequence is shown in SEQ ID NO. 82, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0552] 3) The HLA-CF sequence is shown in SEQ ID NO. 83, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0553] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 84, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0554] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 85, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0555] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 86, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0556] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 87, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0557] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 88, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0558] The upstream and downstream primer sequences of the primer pairs for each site in group 10 are as follows:

[0559] 1) The HLA-AF sequence is shown in SEQ ID NO. 89, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0560] 2) The HLA-BF sequence is shown in SEQ ID NO. 90, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0561] 3) The HLA-CF sequence is shown in SEQ ID NO. 91, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0562] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 92, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0563] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 93, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0564] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 94, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0565] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 95, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0566] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 96, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0567] The upstream and downstream primer sequences of the primer pairs for each site in group 11 are as follows:

[0568] 1) The HLA-AF sequence is shown in SEQ ID NO. 97, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0569] 2) The HLA-BF sequence is shown in SEQ ID NO. 98, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0570] 3) The HLA-CF sequence is shown in SEQ ID NO. 99, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0571] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 100, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0572] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 101, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0573] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 102, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0574] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 103, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0575] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 104, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0576] The upstream and downstream primer sequences of the primer pairs for each site in Group 12 are as follows:

[0577] 1) The HLA-AF sequence is shown in SEQ ID NO. 105, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0578] 2) The HLA-BF sequence is shown in SEQ ID NO. 106, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0579] 3) The HLA-CF sequence is shown in SEQ ID NO. 107, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0580] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 108, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0581] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 109, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0582] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 110, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0583] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 111, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0584] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 112, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0585] The upstream and downstream primer sequences of the primer pairs for each site in Group 13 are as follows:

[0586] 1) The HLA-AF sequence is shown in SEQ ID NO. 113, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0587] 2) The HLA-BF sequence is shown in SEQ ID NO. 114, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0588] 3) The HLA-CF sequence is shown in SEQ ID NO. 115, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0589] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 116, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0590] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 117, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0591] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 118, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0592] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 119, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0593] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 120, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0594] The upstream and downstream primer sequences of the primer pairs for each site in Group 14 are as follows:

[0595] 1) The HLA-AF sequence is shown in SEQ ID NO. 121, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0596] 2) The HLA-BF sequence is shown in SEQ ID NO. 122, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0597] 3) The HLA-CF sequence is shown in SEQ ID NO. 123, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0598] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 124, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0599] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 125, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0600] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 126, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0601] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 127, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0602] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 128, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0603] The upstream and downstream primer sequences of the primer pairs for each site in Group 15 are as follows:

[0604] 1) The HLA-AF sequence is shown in SEQ ID NO. 129, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0605] 2) The HLA-BF sequence is shown in SEQ ID NO. 130, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0606] 3) The HLA-CF sequence is shown in SEQ ID NO. 131, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0607] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 132, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0608] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 133, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0609] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 134, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0610] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 135, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0611] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 136, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0612] The upstream and downstream primer sequences of the primer pairs for each site in Group 16 are as follows:

[0613] 1) The HLA-AF sequence is shown in SEQ ID NO. 137, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0614] 2) The HLA-BF sequence is shown in SEQ ID NO. 138, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0615] 3) The HLA-CF sequence is shown in SEQ ID NO. 139, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0616] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 140, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0617] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 141, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0618] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 142, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0619] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 143, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0620] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 144, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0621] The upstream and downstream primer sequences of the primer pairs for each site in Group 17 are as follows:

[0622] 1) The HLA-AF sequence is shown in SEQ ID NO. 145, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0623] 2) The HLA-BF sequence is shown in SEQ ID NO. 146, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0624] 3) The HLA-CF sequence is shown in SEQ ID NO. 147, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0625] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 148, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0626] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 149, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0627] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 150, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0628] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 151, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0629] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 152, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0630] The upstream and downstream primer sequences of the primer pairs for each site in Group 18 are as follows:

[0631] 1) The HLA-AF sequence is shown in SEQ ID NO. 153, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0632] 2) The HLA-BF sequence is shown in SEQ ID NO. 154, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0633] 3) The HLA-CF sequence is shown in SEQ ID NO. 155, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0634] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 156, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0635] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 157, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0636] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 158, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0637] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 159, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0638] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 160, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0639] The upstream and downstream primer sequences of the primer pairs for each site in Group 19 are as follows:

[0640] 1) The HLA-AF sequence is shown in SEQ ID NO. 161, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0641] 2) The HLA-BF sequence is shown in SEQ ID NO. 162, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0642] 3) The HLA-CF sequence is shown in SEQ ID NO. 163, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0643] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 164, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0644] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 165, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0645] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 166, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0646] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 167, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0647] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 168, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0648] The upstream and downstream primer sequences of the primer pairs for each site in Group 20 are as follows:

[0649] 1) The HLA-AF sequence is shown in SEQ ID NO. 169, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0650] 2) The HLA-BF sequence is shown in SEQ ID NO. 170, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0651] 3) The HLA-CF sequence is shown in SEQ ID NO. 171, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0652] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 172, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0653] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 173, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0654] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 174, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0655] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 175, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0656] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 176, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0657] The upstream and downstream primer sequences of the primer pairs for each site in Group 21 are as follows:

[0658] 1) The HLA-AF sequence is shown in SEQ ID NO. 177, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0659] 2) The HLA-BF sequence is shown in SEQ ID NO. 178, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0660] 3) The HLA-CF sequence is shown in SEQ ID NO. 179, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0661] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 180, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0662] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 181, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0663] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 182, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0664] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 183, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0665] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 184, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0666] The upstream and downstream primer sequences of the primer pairs for each site in Group 22 are as follows:

[0667] 1) The HLA-AF sequence is shown in SEQ ID NO. 185, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0668] 2) The HLA-BF sequence is shown in SEQ ID NO. 186, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0669] 3) The HLA-CF sequence is shown in SEQ ID NO. 187, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0670] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 188, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0671] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 189, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0672] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 190, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0673] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 191, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0674] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 192, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0675] The upstream and downstream primer sequences of the primer pairs for each site in Group 23 are as follows:

[0676] 1) The HLA-AF sequence is shown in SEQ ID NO. 193, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0677] 2) The HLA-BF sequence is shown in SEQ ID NO. 194, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0678] 3) The HLA-CF sequence is shown in SEQ ID NO. 195, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0679] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 196, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0680] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 197, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0681] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 198, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0682] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 199, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0683] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 200, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0684] The upstream and downstream primer sequences of the primer pairs for each site in Group 24 are as follows:

[0685] 1) The HLA-AF sequence is shown in SEQ ID NO. 201, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0686] 2) The HLA-BF sequence is shown in SEQ ID NO. 202, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0687] 3) The HLA-CF sequence is shown in SEQ ID NO. 203, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0688] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 204, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0689] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 205, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0690] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 206, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0691] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 207, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0692] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 208, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0693] The upstream and downstream primer sequences of the primer pairs for each site in group 25 are as follows:

[0694] 1) The HLA-AF sequence is shown in SEQ ID NO. 209, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0695] 2) The HLA-BF sequence is shown in SEQ ID NO. 210, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0696] 3) The HLA-CF sequence is shown in SEQ ID NO. 211, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0697] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 212, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0698] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 213, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0699] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 214, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0700] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 215, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0701] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 216, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0702] The upstream and downstream primer sequences of the primer pairs for each site in Group 26 are as follows:

[0703] 1) The HLA-AF sequence is shown in SEQ ID NO. 217, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0704] 2) The HLA-BF sequence is shown in SEQ ID NO. 218, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0705] 3) The HLA-CF sequence is shown in SEQ ID NO. 219, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0706] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 220, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0707] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 221, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0708] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 222, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0709] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 223, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0710] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 224, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0711] The upstream and downstream primer sequences of the primer pairs for each site in Group 27 are as follows:

[0712] 1) The HLA-AF sequence is shown in SEQ ID NO. 225, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0713] 2) The HLA-BF sequence is shown in SEQ ID NO. 226, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0714] 3) The HLA-CF sequence is shown in SEQ ID NO. 227, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0715] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 228, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0716] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 229, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0717] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 230, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0718] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 231, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0719] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 232, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0720] The upstream and downstream primer sequences of the primer pairs for each site in Group 28 are as follows:

[0721] 1) The HLA-AF sequence is shown in SEQ ID NO. 233, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0722] 2) The HLA-BF sequence is shown in SEQ ID NO. 234, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0723] 3) The HLA-CF sequence is shown in SEQ ID NO. 235, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0724] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 236, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0725] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 237, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0726] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 238, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0727] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 239, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0728] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 240, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0729] The upstream and downstream primer sequences of the primer pairs for each site in Group 29 are as follows:

[0730] 1) The HLA-AF sequence is shown in SEQ ID NO. 241, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0731] 2) The HLA-BF sequence is shown in SEQ ID NO. 242, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0732] 3) The HLA-CF sequence is shown in SEQ ID NO. 243, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0733] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 244, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0734] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 245, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0735] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 246, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0736] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 247, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0737] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 248, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0738] The upstream and downstream primer sequences of the primer pairs for each site in group 30 are as follows:

[0739] 1) The HLA-AF sequence is shown in SEQ ID NO. 249, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0740] 2) The HLA-BF sequence is shown in SEQ ID NO. 250, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0741] 3) The HLA-CF sequence is shown in SEQ ID NO. 251, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0742] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 252, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0743] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 253, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0744] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 254, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0745] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 255, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0746] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 256, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0747] The upstream and downstream primer sequences of the primer pairs for each site in group 31 are as follows:

[0748] 1) The HLA-AF sequence is shown in SEQ ID NO. 257, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0749] 2) The HLA-BF sequence is shown in SEQ ID NO. 258, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0750] 3) The HLA-CF sequence is shown in SEQ ID NO. 259, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0751] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 260, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0752] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 261, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0753] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 262, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0754] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 263, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0755] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 264, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0756] The upstream and downstream primer sequences of the primer pairs for each site in group 32 are as follows:

[0757] 1) The HLA-AF sequence is shown in SEQ ID NO. 265, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0758] 2) The HLA-BF sequence is shown in SEQ ID NO. 266, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0759] 3) The HLA-CF sequence is shown in SEQ ID NO. 267, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0760] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 268, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0761] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 269, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0762] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 270, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0763] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 271, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0764] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 272, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0765] The upstream and downstream primer sequences of the primer pairs for each site in group 33 are as follows:

[0766] 1) The HLA-AF sequence is shown in SEQ ID NO. 273, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0767] 2) The HLA-BF sequence is shown in SEQ ID NO. 274, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0768] 3) The HLA-CF sequence is shown in SEQ ID NO. 275, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0769] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 276, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0770] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 277, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0771] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 278, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0772] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 279, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0773] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 280, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0774] The upstream and downstream primer sequences of the primer pairs for each site in group 34 are as follows:

[0775] 1) The HLA-AF sequence is shown in SEQ ID NO. 281, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0776] 2) The HLA-BF sequence is shown in SEQ ID NO. 282, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0777] 3) The HLA-CF sequence is shown in SEQ ID NO. 283, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0778] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 284, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0779] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 285, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0780] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 286, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0781] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 287, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0782] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 288, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0783] The upstream and downstream primer sequences of the primer pairs for each site in group 35 are as follows:

[0784] 1) The HLA-AF sequence is shown in SEQ ID NO. 289, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0785] 2) The HLA-BF sequence is shown in SEQ ID NO. 290, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0786] 3) The HLA-CF sequence is shown in SEQ ID NO. 291, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0787] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 292, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0788] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 293, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0789] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 294, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0790] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 295, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0791] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 296, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0792] The upstream and downstream primer sequences of the primer pairs for each site in group 36 are as follows:

[0793] 1) The HLA-AF sequence is shown in SEQ ID NO. 297, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0794] 2) The HLA-BF sequence is shown in SEQ ID NO. 298, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0795] 3) The HLA-CF sequence is shown in SEQ ID NO. 299, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0796] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 300, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0797] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 301, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0798] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 302, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0799] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 303, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0800] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 304, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0801] The upstream and downstream primer sequences of the primer pairs for each site in group 37 are as follows:

[0802] 1) The HLA-AF sequence is shown in SEQ ID NO. 305, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0803] 2) The HLA-BF sequence is shown in SEQ ID NO. 306, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0804] 3) The HLA-CF sequence is shown in SEQ ID NO. 307, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0805] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 308, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0806] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 309, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0807] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 310, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0808] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 311, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0809] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 312, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0810] The upstream and downstream primer sequences of the primer pairs for each site in group 38 are as follows:

[0811] 1) The HLA-AF sequence is shown in SEQ ID NO. 313, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0812] 2) The HLA-BF sequence is shown in SEQ ID NO. 314, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0813] 3) The HLA-CF sequence is shown in SEQ ID NO. 315, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0814] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 316, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0815] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 317, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0816] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 318, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0817] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 319, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0818] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 320, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0819] The upstream and downstream primer sequences of the primer pairs for each site in group 39 are as follows:

[0820] 1) The HLA-AF sequence is shown in SEQ ID NO. 321, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0821] 2) The HLA-BF sequence is shown in SEQ ID NO. 322, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0822] 3) The HLA-CF sequence is shown in SEQ ID NO. 323, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0823] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 324, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0824] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 325, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0825] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 326, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0826] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 327, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0827] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 328, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0828] The upstream and downstream primer sequences of the primer pairs for each site in group 40 are as follows:

[0829] 1) The HLA-AF sequence is shown in SEQ ID NO. 329, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0830] 2) The HLA-BF sequence is shown in SEQ ID NO. 330, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0831] 3) The HLA-CF sequence is shown in SEQ ID NO. 331, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0832] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 332, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0833] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 333, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0834] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 334, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0835] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 335, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0836] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 336, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0837] The upstream and downstream primer sequences of the primer pairs for each site in group 41 are as follows:

[0838] 1) The HLA-AF sequence is shown in SEQ ID NO. 337, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0839] 2) The HLA-BF sequence is shown in SEQ ID NO. 338, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0840] 3) The HLA-CF sequence is shown in SEQ ID NO. 339, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0841] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 340, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0842] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 341, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0843] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 342, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0844] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 343, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0845] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 344, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0846] The upstream and downstream primer sequences of the primer pairs for each site in group 42 are as follows:

[0847] 1) The HLA-AF sequence is shown in SEQ ID NO. 345, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0848] 2) The HLA-BF sequence is shown in SEQ ID NO. 346, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0849] 3) The HLA-CF sequence is shown in SEQ ID NO. 347, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0850] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 348, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0851] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 349, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0852] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 350, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0853] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 351, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0854] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 352, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0855] The upstream and downstream primer sequences of the primer pairs for each site in group 43 are as follows:

[0856] 1) The HLA-AF sequence is shown in SEQ ID NO. 353, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0857] 2) The HLA-BF sequence is shown in SEQ ID NO. 354, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0858] 3) The HLA-CF sequence is shown in SEQ ID NO. 355, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0859] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 356, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0860] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 357, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0861] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 358, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0862] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 359, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0863] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 360, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0864] The upstream and downstream primer sequences of the primer pairs for each site in group 44 are as follows:

[0865] 1) The HLA-AF sequence is shown in SEQ ID NO. 361, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0866] 2) The HLA-BF sequence is shown in SEQ ID NO. 362, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0867] 3) The HLA-CF sequence is shown in SEQ ID NO. 363, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0868] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 364, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0869] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 365, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0870] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 366, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0871] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 367, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0872] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 368, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0873] The upstream and downstream primer sequences of the primer pairs for each site in group 45 are as follows:

[0874] 1) The HLA-AF sequence is shown in SEQ ID NO. 369, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0875] 2) The HLA-BF sequence is shown in SEQ ID NO. 370, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0876] 3) The HLA-CF sequence is shown in SEQ ID NO. 371, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0877] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 372, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0878] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 373, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0879] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 374, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0880] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 375, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0881] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 376, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0882] The upstream and downstream primer sequences of the primer pairs for each site in group 46 are as follows:

[0883] 1) The HLA-AF sequence is shown in SEQ ID NO. 377, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0884] 2) The HLA-BF sequence is shown in SEQ ID NO. 378, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0885] 3) The HLA-CF sequence is shown in SEQ ID NO. 379, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0886] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 380, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0887] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 381, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0888] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 382, ​​and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0889] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 383, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0890] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 384, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0891] The upstream and downstream primer sequences of the primer pairs for each site in group 47 are as follows:

[0892] 1) The HLA-AF sequence is shown in SEQ ID NO. 385, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0893] 2) The HLA-BF sequence is shown in SEQ ID NO. 386, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0894] 3) The HLA-CF sequence is shown in SEQ ID NO. 387, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0895] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 388, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0896] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 389, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0897] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 390, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0898] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 391, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0899] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 392, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1;

[0900] The upstream and downstream primer sequences of the primer pairs for each site in group 48 are as follows:

[0901] 1) The HLA-AF sequence is shown in SEQ ID NO. 393, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1;

[0902] 2) The HLA-BF sequence is shown in SEQ ID NO. 394, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1;

[0903] 3) The HLA-CF sequence is shown in SEQ ID NO. 395, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1;

[0904] 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 396, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1;

[0905] 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 397, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1;

[0906] 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 398, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1;

[0907] 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 399, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1;

[0908] 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 400, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in Group 1.

[0909] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After sequence synthesis, quality control and quantification were performed, and then diluted to the required concentration as needed.

[0910] The above primer set can be used to prepare an HLA ultra-high-resolution genotyping detection kit, which also contains an HLA gene rapid amplification enzyme system Flash PCR Master Mix For Long Fragment.

[0911] When testing one sample, you can select one of the 48 primer sets and perform the test according to the following method. When testing two samples, you can select two of the 48 primer sets and perform the test according to the following method. And so on. Up to 11 gene loci of 48 different samples can be tested simultaneously. If necessary, the number of barcode types can be further expanded to allow more samples to be tested simultaneously in a single batch.

[0912] 2. Target gene amplification

[0913] 1. Primer dilution and mixing

[0914] The 24 HLA primers were diluted to 100 μM with low-TE (10 mM Tris, 1 mM EDTA, pH 8.0) and the following ratios were used: HLA-AF:HLA-A-R1:HLA-A-R2:HLA-BF:HLA-B-R1:HLA-B-R2:HLA-CF:HLA-C-R1:HLA-C-R2:HLA-DPA-F1:HLA-DPA-F2:HLA-DPA-R:HLA-DPB-F:HLA-DPB-R1:HLA-DPB-R2: HLA-DQA-F:HLA-DQA-R1:HLA-DQA-R2:HLA-DQB-F:HLA-DQB-R1:HLA-DQB-R2:HLA-DRB1345-F:HLA-DR B1345-R1:HLA-DRB1345-R2=10:7:7:10:7:7:10:7:7:6:6:10:10:6:6:10:7:7:10:6:6:15:8:8 for mixing.

[0915] 2. Amplification system

[0916] Table 3. Reaction system of group 1

[0917] Reagents Volume (ul) FlashPCRMasterMixForLongFragment(CW3397M) 12.5 Primers 1 RNase-Free Water (CW0612M) 11.5-x DNA (30ng) x Total volume 25

[0918] Prepare the long fragment amplification system according to Table 3 in a PCR tube on an ice box, add 30 ng of DNA template, pipette to mix, and centrifuge briefly.

[0919] 3. Amplification Procedure

[0920] Place the PCR tube into the PCR instrument and perform the reaction according to the conditions in Table 4.

[0921] Table 4. Long fragment amplification program

[0922]

[0923] 3. Purification, Quantification, and Fragment Analysis of PCR Amplification Products

[0924] 1. PCR Product Purification

[0925] (1) Preparation of reagents and consumables: Take out the Kangwei Century DNA purification magnetic beads (Cat. No.: CW3171M) stored at 4°C and equilibrate them at room temperature for about 30 minutes; prepare 80% alcohol and prepare 440uL / reaction.

[0926] (2) After the magnetic beads have equilibrated to room temperature, mix them using a vortex shaker at medium speed for 1 minute. Add 12.5 μl (0.5X) of the magnetic bead suspension to the PCR tube. Mix by pipetting at half scale approximately 10 times until the beads are evenly mixed.

[0927] (3) Incubate at room temperature for 5 minutes.

[0928] (4) After incubation, place the PCR tube on a magnetic rack for about 3 minutes. When the solution becomes clear, discard the supernatant completely, taking care not to let the tip of the pipette touch the magnetic beads.

[0929] (5) Keep the PCR tubes on the magnetic rack, add 200 μl of freshly prepared 80% alcohol to each tube, and let it stand at room temperature for about 30 seconds.

[0930] (6) When the solution becomes clear, remove the supernatant, taking care not to let the tip of the pipette touch the magnetic beads.

[0931] (7) Repeat steps (5) and (6) once for a second wash.

[0932] (8) Keep the PCR tube fixed on the magnetic rack, open the tube cap, and dry it at room temperature for 3 to 5 minutes until the magnetic beads are non-reflective and cracked.

[0933] (9) Remove the PCR tube from the magnetic stand, add 35 μL of deionized water to each well, pipette to mix, incubate at room temperature for about 10 minutes, and centrifuge briefly.

[0934] (10) Place the PCR tube on a magnetic rack. When the solution becomes clear, transfer about 30 μL of the purified amplified product to a new PCR tube. Be careful not to let the tip of the pipette touch the magnetic beads.

[0935] 2. PCR Product Quantification

[0936] Quantitative use of PCR products dsDNA BR Assay Kits (Cat. No.: Q32853), Qubit TM Test tubes (Cat. No. Q32856) and Qubit TM 4. Use a fluorometer (Cat. No. Q33238) to quantify the product according to the standard procedure in the instructions and record the product concentration;

[0937] 3. PCR Product Fragment Analysis

[0938] Fragment analysis of PCR products was performed on an Agilent 4200 TapeStation capillary electrophoresis analyzer and associated reagents.

[0939] IV. Construction of the third-generation HLA library

[0940] 1. PCR product mixing

[0941] Mix equal amounts of PCR amplification products by mass in a 0.2 ml PCR tube. The total amount of amplicon sample input is 2000 ng (for example, for 20 samples, 100 ng of template can be added per sample). If the number of samples is less than 2000 ng, add all of them. Mix gently, centrifuge briefly, and collect the reaction solution at the bottom of the tube. Ensure that there are no bubbles in the reaction solution.

[0942] 2. End repair

[0943] (1) ERAT Mix (CW3045) and 10× ERAT Buffer (CW3045) are the enzymes and buffers for end-repair and +A, respectively. Prepare the end-repair system on ice according to Table 5 and dispense into PCR tubes. Then, add ≥2000 ng of PCR product to the PCR tubes, gently vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube, ensuring that there are no bubbles in the reaction solution.

[0944] Table 5. End repair reaction system

[0945] Components Volume (μL) Pooling PCR products X Nuclease-free water 21.5-X 10×ERAT Buffer (CW3045) 2.5 ERATMix(CW3045) 1 Total volume 25

[0946] (2) Place the above PCR tube into a PCR instrument and perform the reaction according to Table 6.

[0947] Table 6. End repair reaction conditions

[0948]

[0949] (3) After the reaction is completed, proceed directly to the next step.

[0950] 3. Sequencing adapter ligation

[0951] (1) Prepare the mixture according to Table 7 in a 0.2 mL PCR tube, mix gently, and then centrifuge briefly to collect the reaction solution at the bottom of the tube, ensuring that there are no bubbles in the reaction solution.

[0952] Table 7. Linker ligation reaction solution system

[0953] Components Volume (μl) End repair products 25 Hydration 5.5 LA connector 4 T4 DNA ligase Buffer (CW2701) 12 T4 DNA ligase, 15 U / μL (CW2701) 3.5 Total volume 50

[0954] (2) Place the PCR tube containing the reaction mixture from the previous step on a PCR instrument and react according to the conditions in Table 8:

[0955] Table 8. Adapter ligation reaction conditions

[0956]

[0957] 4. Magnetic Bead Purification

[0958] (1) Equilibrate the magnetic beads to room temperature in advance, add the reaction system to 25 μL (0.5X) of Kangwei Century DNA purification magnetic beads (1.5 mL centrifuge tube), and gently mix by flicking or pipetting; incubate at room temperature for 5 minutes;

[0959] (2) Transfer the test tube to the magnetic rack and let it stand for 2-5 minutes until the liquid becomes clear. Carefully discard the supernatant (be careful not to touch the magnetic beads);

[0960] (3) Remove the EP tube from the magnetic rack, slowly add 125 μL of SFB (short fragment buffer, SFB) along the side where the magnetic beads are adsorbed, rotate the EP tube to gently resuspend the magnetic beads, and then put it back on the magnetic rack (the magnetic beads are not attached to the wall to form aggregates). After the liquid becomes clear, aspirate the clear liquid and be careful not to touch the magnetic beads);

[0961] (4) Repeat the previous step (using SFB for secondary cleaning and impurity removal);

[0962] (5) Remove the EP tube and centrifuge it briefly using a mini centrifuge. Carefully discard the remaining liquid with a pipette, open the tube cap, and air dry for 30 seconds (do not allow it to dry out and crack).

[0963] (6) Add 35 μL of Elution Buffer (EB) to elute the nucleic acid, flick the tube to mix, and incubate at 37°C for 10 min (tap every 2 minutes);

[0964] (7) Place the EP tube back on the magnetic rack until the magnetic beads are attracted to one side of the magnetic rack or the mixed solution becomes clear. Transfer 32 μL of the supernatant to a clean PCR tube.

[0965] 5. Library Quantification

[0966] (1) Take 1 μL of DNA library and perform Qubit fluorescence quantification. Record the concentration.

[0967] (2) Take 150ng of library for experiment.

[0968] 6. Sequencing

[0969] The sequencing platform was the PromethlON 2Solo nanopore sequencer from Oxford Nanopore Technologies (ONT).

[0970] 1. Chip loading and sequencing

[0971] (1) Reagent preparation: Take out Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) from the refrigerator, melt them, vortex mix, and place on ice.

[0972] (2) Use a 1.5 ml centrifuge tube to prepare the reagents (priming mix) according to the system in Table 9:

[0973] Table 9. Primingmix system configuration

[0974]

[0975] (3) Mix the priming mix by pipetting and adding 800 μl of priming mix through the flow cell priming port (before adding liquid, adjust the 1 ml pipette to 200 μl, insert the pipette tip into the flow cell priming port, and aspirate back to 220-230 μl);

[0976] (4) Let it stand for 5 minutes;

[0977] (5) Use a 1.5 ml centrifuge tube to prepare the library for loading system according to Table 10:

[0978] Table 10. Library for loading system configuration

[0979] Reagents volume SequencingBuffer (SB) 37.5μl LibraryBeads (LIB) (mix by pipetting before aspiration) 25.5μl DNA library X μl (approximately 15 fmol, can be higher if the sample size is small) EB 12-X Total 75 μl

[0980] (6) Add 200 μl of priming mix through the flow cell priming port (before adding liquid, adjust the 1 ml pipette to 200 μl, insert the pipette tip into the flow cell priming port, and aspirate back to 220-230 μl);

[0981] (7) Add 75 μl of library loading reagent through the SpotON sample port (mix thoroughly before adding and drip at a constant rate).

[0982] (8) Cover the SpotON sample port and the flow cell priming port;

[0983] (9) Start sequencing (pay attention to the kit number).

[0984] 2. Clean the chip

[0985] (1) Take out Wash Mix (WMX), Wash Diluent (DIL), and Storage Buffer (S) and thaw them at room temperature. Wash Mix (WMX) can be mixed by inversion, but not vortexing. Wash Diluent (DIL) and Storage Buffer (S) can be mixed by vortexing.

[0986] (2) Prepare Flow Cell Wash Mix according to Table 11.

[0987] Table 11. Flow Cell Wash Mix

[0988]

[0989]

[0990] (3) Mix thoroughly with a pipette and place on ice;

[0991] (4) Open the flow cell priming port (before adding liquid, adjust the 1 ml pipette to 200 μl, insert the pipette tip into the flow cell priming port, and aspirate back to 220-230 μl), and add 400 μl Flow Cell Wash Mix at a constant speed;

[0992] (5) Let stand at room temperature for 30 minutes;

[0993] (6) Open the flow cell priming port (before adding liquid, adjust the 1 ml pipette to 200 μl, insert the pipette tip into the flow cell priming port, and aspirate back to 220-230 μl), and add 500 μl Storage Buffer (S) at a constant speed;

[0994] (7) Cover the priming port and test the number of remaining available holes;

[0995] (8) sucking waste liquid from the waste liquid port and discarding it;

[0996] (9) Mark the chip with the expiration date and the number of remaining available wells, and store it at 4°C.

[0997] 7. Data Analysis

[0998] Sequencing data were converted to the fastq format using the dorado analysis module and segmented according to barcode information to generate sequencing data corresponding to each sample. The sequencing data were then analyzed using CWBIO-LINKAGE software from Cosmos to determine the typing results for the HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci.

[0999] 8. Results

[1000] 1. The results of the 11-site PCR amplification product analysis of HLA in 3 clinical blood samples are shown in Figure 2 The amplicon fragments were all larger than 4000 bp. Overall, the amplified fragments were mainly concentrated between 5000 bp and 10000 bp. The fragment sizes were in line with expectations, and the overall uniformity of the bands was relatively good.

[1001] 2. The nanopore-based full-length HLA gene sequencing method established in this example was used to test 30 national reference samples. Ultra-high-resolution typing results were obtained for all 11 loci of the 30 national reference samples (see Table 12), and the consistency rate with the national reference standard typing results was 100%.

[1002] Table 12. Test results of HLA national reference products

[1003]

[1004]

[1005]

[1006]

[1007]

[1008]

[1009]

[1010]

[1011] 3. 28 clinical blood samples were tested using the embodiment, and high-resolution HLA typing results were obtained for all samples using the patented method (see Table 13). Among them, a new allele HLA-A*24:NEW was identified in the HLA-S2 sample.

[1012] Table 13. HLA analysis results of 28 clinical blood samples

[1013]

[1014]

[1015]

[1016] Example 2

[1017] Primer Design and Synthesis HLA-related gene sequences were searched on NCBI (National Center of Biotechnology Information). Based on the primer design principles for multiplex PCR, specific primer sequences and index sequences were designed (see Tables 14 and 15 below).

[1018] Table 14 Specific primer sequences

[1019]

[1020]

[1021] Table 15 Index synthesis related oligonucleotide sequences

[1022]

[1023]

[1024]

[1025]

[1026]

[1027]

[1028]

[1029]

[1030] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After sequence synthesis, the sequences were quality-checked and quantified, and then diluted to the desired concentration as needed. The present invention provides 48 index sequences that can be used to distinguish 48 different samples during sequencing.

[1031] The primer set in Table 14 can be used to prepare an HLA ultra-high-resolution genotyping detection kit. The kit also contains an HLA gene rapid amplification enzyme system Flash PCR Master Mix For Long Fragment and the Index synthesis-related oligonucleotide sequences in Table 15.

[1032] When testing a single sample, you can select one of the 48 indexes for labeling and perform the test as follows. When testing two samples, you can select two of the 48 indexes for labeling and perform the test as follows. This process can be repeated in this way, allowing you to label up to 48 different samples simultaneously. If needed, you can expand the number of index types to allow for simultaneous testing of more samples in a single batch.

[1033] 1. Preparation of double-stranded nucleic acid for index sample label ligation

[1034] 1. Preparation of 100uM stock solution

[1035] 1) Centrifuge the EP tube containing the oligonucleotide powder at 12000 rpm for 2 minutes;

[1036] 2) Add the appropriate volume of enzyme-free water to the dry powder according to the order feedback information and dilute it to a 100uM stock solution.

[1037] 3) Take 1 μl of the stock solution for nanodrop quantification and calculate the actual molar concentration. If it is <100 μM or >130 μM, subsequent electrophoresis and library quality control are required.

[1038] 4) Record A260 / 280 and A260 / 230. If A260 / 280 < 1.6 or A260 / 280 > 2.2, subsequent electrophoresis and library quality control are required.

[1039] 2. Preparation of 10uM working solution

[1040] 1) Prepare the working fluid according to the following table.

[1041] Table 16 Final concentration 10uM preparation system

[1042] Reagents 25ul system addition amount (ul) 50ul system addition amount (ul) Final concentration 4xT4DANLligasebuffer 6.25 12.5 1X indexF(100uM) 2.5 5 10uM indexR(100uM) 2.5 5 10uM T4PNK (10u / ul) 1.0 2.0 ddH2O 12.75 25.5 Total 25 50

[1043] 2) Set up the annealing program

[1044] Annealing procedure: 37℃ / 30min,

[1045] 95℃ / 2min、0.1℃ / s to 90℃,

[1046] 90℃ / 2min, 0.1℃ / s to 85℃,

[1047] ...,

[1048] 35℃ / 2min、0.1℃ / s to 30℃,

[1049] 30℃ / 2min, 0.1℃ / s to 25℃,

[1050] 25℃Hold.

[1051] 3) Annealed adapter quality inspection - 2% agarose gel electrophoresis

[1052] Reagents: 6× SuperStain Loading Buffer (CW2636); SuperDNA Marker (CW2583)

[1053] Sample volume 1ul, 6×SuperStain Loading Buffer volume 1ul, ddH2O volume 4ul, a total of 6ul, sample application;

[1054] Marker 5ul, 6× SuperStain Loading Buffer 1ul, a total of 6ul, sample application;

[1055] Qualified standard: If the connector annealing is qualified, there will be only one main band. If there are miscellaneous bands, it is unusable and needs to check the sample purity or re-anneal. Figure 3 .

[1056] 2. Target gene amplification

[1057] 1. Primer dilution and mixing

[1058] The primers for the 24 HLAs were diluted to 100 μM with low-TE (10 mM Tris, 1 mM EDTA, pH 8.0) and the following ratios were used: HLA-A_F_1:HLA-A_R_1:HLA-B_F_1:HLA-B_R_1:HLA-C_F_1:HLA-C_R_1:HLA-DPA_F_1:HLA-DPA_R_1:HLA-DPB_F_1:HLA-DPB_R_1:HLA-DQA_F_1:HLA-DQA_R_1:HLA-DQB_F_1:HLA-DQB_R_1:HLA-DRB1345_F_1:HLA-DRB1345_R_1:HLA-A_F_2:HLA-A_R_2:HLA-B_F_2:HLA LA-B_R_2:HLA-C_F_2:HLA-C_R_2:HLA-DPA_F_2:HLA-DPA_R_2:HLA-DPB_F_2:HLA-DPB_R_2:HLA-DQA_F_2:HLA-DQA_R_2:HLA-DQB_F_2:HLA-DQB_R_2:HLA-DRB1345_F_2:HLA-DRB1345_R_2=1:1:1:1:2:2::1:1:2:2:1:1:2:2:4:4:2:2:1:1:2:2:1:1:3:3:2:2:2:2:2:4 were mixed and diluted 5 times and PCR process was performed according to the following amplification system.

[1059] Amplification system

[1060] Table 17. Reaction system of group 1

[1061]

[1062]

[1063] Prepare the long fragment amplification system according to Table 3 in a PCR tube on an ice box, add 30 ng of DNA template, pipette to mix, and centrifuge briefly.

[1064] Amplification procedure

[1065] Place the PCR tube into the PCR instrument and perform the reaction according to the conditions in Table 4.

[1066] Table 18. Long fragment amplification program

[1067]

[1068] 2. Purification of PCR Amplification Products

[1069] ① Preparation of reagents and consumables: Take out the Kangwei Century DNA purification magnetic beads (Cat. No.: CW3171M) stored at 4°C and equilibrate them at room temperature for about 30 minutes; prepare 80% alcohol and prepare 440uL / reaction.

[1070] ② After the magnetic beads have equilibrated to room temperature, mix them using a vortex shaker at medium speed for 1 minute. Add 12.5 μl (0.5X) of magnetic bead suspension to the PCR tube. Mix by pipetting at half-scale about 10 times until the beads are evenly mixed.

[1071] ③Incubate at room temperature for 5 minutes.

[1072] ④After incubation, place the PCR tube on the magnetic stand for about 3 minutes. When the solution becomes clear, discard the supernatant completely, taking care not to let the pipette tip touch the magnetic beads.

[1073] ⑤ Keep the PCR tubes on the magnetic rack, add 200 μl of freshly prepared 80% alcohol to each tube, and let it stand at room temperature for about 30 seconds.

[1074] ⑥ When the solution becomes clear, remove the supernatant, taking care not to let the tip of the pipette touch the magnetic beads.

[1075] ⑦ Repeat steps (5) and (6) once for the second washing.

[1076] ⑧ Keep the PCR tube fixed on the magnetic rack, open the tube cap, and dry at room temperature for 3-5 minutes until the magnetic beads are non-reflective and have not cracked. ⑨ Remove the PCR tube from the magnetic rack and add 35uL of deionized water to each well. Pipet and mix thoroughly. Incubate at room temperature for approximately 10 minutes and briefly centrifuge.

[1077] ⑩ Place the PCR tube on the magnetic rack. When the solution becomes clear, transfer about 30uL of the purified amplification product to a new PCR tube. Be careful not to let the tip of the pipette touch the magnetic beads.

[1078] The purified PCR product was quantified using qubit2.0 and placed on ice for later use.

[1079] 3. End Repair

[1080] 1. Prepare the final reaction system, see Table 4 for details.

[1081] Table 19 Preparation of end repair reaction solution

[1082] Components Volume (μl) - 1 reaction PCR product samples X 10×ERAT Buffer (CW3045) 2.5 ERATMix(CW3045) 1 Nuclease-free water 21.5-X Total volume 25

[1083] Place the PCR tube containing the reaction mixture from the previous step on a PCR instrument and react according to the following conditions (Table 5):

[1084] Table 20 End repair reaction conditions

[1085]

[1086] Four index connections

[1087] 1. Connection system configuration

[1088] Add the corresponding index to each sample and record it (Cwbar0001-1343). Prepare the mixture in a 0.2mL PCR tube. Gently mix, then briefly centrifuge to collect the reaction solution at the bottom of the tube, ensuring there are no bubbles. The reaction system is as follows (when preparing a mix or adding a single addition, add the enzyme last):

[1089] Table 21 Preparation of index ligation reaction solution

[1090] Components Volume (μl) - 1 reaction End-repaired DNA 14.25 Nativeindex 2.5 4XT4 DNA ligase Buffer (CW2701) 6.25 T4 DNA ligase (CW2701) 2 Total volume 25

[1091] Note: Samples and indexes need to correspond one to one. When adding indexes, contamination must be prevented.

[1092] Place the PCR tube containing the reaction mixture from the previous step on a PCR instrument and react according to the following conditions:

[1093] Table 22 Index ligation reaction conditions

[1094] temperature time Hot cover (off) 23℃ / room temperature 15min

[1095] Note: After this step, proceed to the next step immediately.

[1096] 2. Magnetic Bead Purification

[1097] 1) Take out the magnetic beads in advance, mix them, and equilibrate them at room temperature for 30 minutes. According to the amount of subsequent mixed samples, take the corresponding volume of magnetic beads in advance and divide them into 8 strips for later use (the magnetic beads can be divided into 8 strips according to the amount of experimental samples).

[1098] 2) Sample mixing: Take an equal amount of the reaction product from the previous step and add it to the eight pre-packed strips of magnetic beads. Then mix the eight strips of 0.5X magnetic beads with samples into the same tube. Terminate the ligation reaction at the same time, mix well, and incubate at room temperature for 5 minutes.

[1099] 3) From the incubated mixed sample with magnetic beads, take 1 mL of the mixed sample and transfer it to a new EP tube for purification. If the total volume is less than 1 mL, purify the entire volume. Transfer the EP tube to the magnetic rack and let it stand for about 2-5 minutes. When the liquid becomes clear, carefully discard the supernatant (be careful not to contact the magnetic beads).

[1100] 4) Maintaining the magnetic attraction, add 1 mL of 80% ethanol to the tube (enough to cover the magnetic beads). Do not pipette directly onto the beads. After a moment, carefully discard the supernatant (taking care not to touch the magnetic beads).

[1101] 5) Repeat the previous step (using 80% ethanol to wash twice to remove impurities);

[1102] 6) Remove the EP tube, spin it away instantly, and carefully discard the remaining liquid with a pipette; open the tube cap and let the magnetic beads dry for about 3-5 minutes until the beads have no reflective surface (the beads should not be dry and cracked);

[1103] 7) Add 33 μl of nuclease-free water to elute the nucleic acid, and incubate in a constant temperature shaking metal bath at 300 rpm and 37°C for 5 min.

[1104] 8) Return the tube to the magnetic rack and incubate until the beads are attracted to one side of the rack or the mixture becomes clear. Transfer 32 μl of the supernatant to a clean PCR tube.

[1105] 9) Take 1 μL of the purified product for Qubit quantification.

[1106] 10) Take 300 ng of the purified product for the next step of sequencing linker ligation experiment.

[1107] 3. Sequencing adapter ligation

[1108] 3.1 System configuration

[1109] Prepare the mixture in a 0.2 mL PCR tube, mix gently, and then centrifuge briefly to collect the reaction solution at the bottom of the tube, ensuring that there are no bubbles in the reaction solution. The reaction system is as follows:

[1110] Table 23 Preparation of adapter ligation reaction solution

[1111] Components Volume (μl) Purified product from the previous step 30 Sequencing adapters (NA) 2.5 4XT4 DNA ligase Buffer (CW2701) 12.5 T4 DNA ligase, 15 U / μL (CW2701) 5 EB Fill volume to 50

[1112] 3.2 Reaction Conditions Place the PCR tube containing the reaction mixture from the previous step on a PCR instrument and react according to the following conditions:

[1113] Table 24 Connector ligation reaction conditions

[1114]

[1115]

[1116] 3.3 Magnetic bead purification

[1117] 1) Remove the magnetic beads in advance, mix well, and equilibrate at room temperature for 30 minutes. Add the sample to the magnetic beads (1.5 mL centrifuge tube, 0.5X magnetic bead purification), gently mix by flicking or pipetting, and incubate at 37°C for 5 minutes.

[1118] 2) Transfer the centrifuge tube to a magnetic rack and let it sit for about 2-5 minutes. When the liquid becomes clear, carefully discard the supernatant (be careful not to touch the magnetic beads).

[1119] 3) Remove the EP tube from the magnetic stand, add 125 μl SFB (short fragment buffer (SFB), remove the centrifuge tube from the magnetic stand, flick to mix the magnetic beads, and return it to the magnetic stand. When the liquid becomes clear, aspirate the supernatant (be careful not to touch the magnetic beads);

[1120] 4) Repeat the previous step (using SFB for secondary cleaning and impurity removal);

[1121] 5) Remove the centrifuge tube from the magnetic rack, spin briefly, carefully discard any remaining liquid with a pipette, open the tube cap, and air-dry for 30 seconds.

[1122] 6) Add 33 μl Elution Buffer (EB) to elute nucleic acids, flick to mix, and incubate at 37°C, 300 rpm, for 8 min.

[1123] 7) Place the EP tube back on the magnetic stand until the magnetic beads are attracted to one side of the magnetic stand or the mixture becomes clear. Pipette 32 μl of the supernatant and transfer it to a clean PCR tube.

[1124] 8) Take 1 μL of purified product for Qubit quantification (if the input amount is 300 ng, the Qubit concentration should be ≥ 2.5 ng / ul).

[1125] 9) Take 20 ng of the purified product for the experiment.

[1126] Note: Do not clean the chip immediately after sequencing is completed. If the bioinformatics feedback indicates insufficient data and the sequencing is unsuccessful, you can continue to click Start Sequencing for additional testing.

[1127] 3.4 Library Quality Control

[1128] After purification, the library was subjected to electrophoresis quality inspection, and the results are shown in Figure 4 , the library size and distribution are consistent with the expected product size distribution, and the library continues to proceed with sequencing adapter ligation and upper-level sequencing.

[1129] 5. Sequencing

[1130] Refer to the process of Example 1. The blood samples and reference samples tested in Example 2 are the same as those in Example 1, and the typing results are completely consistent.

[1131] The above are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A primer set for HLA ultra-high resolution genotyping, wherein the primer set is used for HLA gene multiplex amplification, characterized in that: The primer set includes primer pairs for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci, and the upstream and downstream primers of the primer pairs and the related nucleotide sequences are selected from any one or more of the following groups 1 to 48: The upstream and downstream primer sequences of the primer pairs for each site in Group 1 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 1, the HLA-A-R1 sequence is shown in SEQ ID NO. 2, and the HLA-A-R2 sequence is shown in SEQ ID NO. 3; 2) The HLA-BF sequence is shown in SEQ ID NO. 4, the HLA-B-R1 sequence is shown in SEQ ID NO. 5, and the HLA-B-R2 sequence is shown in SEQ ID NO. 6; 3) The HLA-CF sequence is shown in SEQ ID NO. 7, the HLA-C-R1 sequence is shown in SEQ ID NO. 8, and the HLA-C-R2 sequence is shown in SEQ ID NO. 9; 4) The HLA-DPA-F1 sequence is shown in SEQ ID NO. 10, the HLA-DPA-F2 sequence is shown in SEQ ID NO. 11, and the HLA-DPA-R sequence is shown in SEQ ID NO. 12; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 13, the HLA-DPB-R1 sequence is shown in SEQ ID NO. 14, and the HLA-DPB-R2 sequence is shown in SEQ ID NO. 15; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 16, the HLA-DQA-R1 sequence is shown in SEQ ID NO. 17, and the HLA-DQA-R2 sequence is shown in SEQ ID NO. 18; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 19, the HLA-DQB-R1 sequence is shown in SEQ ID NO. 20, and the HLA-DQB-R2 sequence is shown in SEQ ID NO. 21; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 22, the HLA-DRB1345-R1 sequence is shown in SEQ ID NO. 23, and the HLA-DRB1345-R2 sequence is shown in SEQ ID NO. 24; The upstream and downstream primer sequences of the primer pairs for each site in Group 2 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 25, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 26, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 27, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 28, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 29, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 30, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 31, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 32, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 3 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 33, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 34, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 35, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 36, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 37, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 38, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 39, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 40, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 4 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 41, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 42, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 43, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 44, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 45, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 46, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 47, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 48, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 5 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 49, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 50, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 51, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 52, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 53, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 54, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 55, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 56, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 6 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 57, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 58, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 59, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 60, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 61, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 62, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 63, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 64, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 7 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 65, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 66, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 67, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 68, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 69, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 70, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 71, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 72, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 8 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 73, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 74, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 75, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 76, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 77, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 78, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 79, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 80, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 9 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 81, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 82, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 83, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 84, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 85, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 86, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 87, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 88, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 10 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 89, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 90, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 91, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 92, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 93, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 94, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 95, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 96, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 11 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 97, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 98, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 99, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 100, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 101, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 102, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 103, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 104, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 12 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 105, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 106, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 107, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 108, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 109, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 110, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 111, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 112, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 13 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 113, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 114, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 115, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 116, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 117, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 118, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 119, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 120, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 14 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 121, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 122, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 123, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 124, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 125, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 126, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 127, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 128, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 15 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 129, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 130, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 131, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 132, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 133, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 134, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 135, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 136, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 16 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 137, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 138, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 139, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 140, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 141, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 142, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 143, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 144, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 17 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 145, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 146, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 147, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 148, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 149, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 150, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 151, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 152, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 18 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 153, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 154, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 155, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 156, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 157, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 158, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 159, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 160, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 19 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 161, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 162, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 163, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 164, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 165, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 166, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 167, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 168, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 20 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 169, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 170, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 171, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 172, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 173, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 174, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 175, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 176, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 21 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 177, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 178, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 179, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 180, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 181, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 182, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 183, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 184, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 22 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 185, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 186, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 187, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 188, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 189, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 190, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 191, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 192, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 23 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 193, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 194, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 195, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 196, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 197, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 198, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 199, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 200, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 24 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 201, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 202, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 203, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 204, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 205, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 206, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 207, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 208, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 25 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 209, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 210, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 211, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 212, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 213, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 214, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 215, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 216, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 26 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 217, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 218, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 219, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 220, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 221, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 222, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 223, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 224, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 27 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 225, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 226, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 227, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 228, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 229, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 230, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 231, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 232, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 28 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 233, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 234, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 235, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 236, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 237, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 238, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 239, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 240, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in Group 29 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 241, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 242, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 243, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 244, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 245, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 246, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 247, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 248, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 30 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 249, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 250, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 251, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 252, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 253, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 254, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 255, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 256, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 31 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 257, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 258, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 259, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 260, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 261, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 262, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 263, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 264, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 32 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 265, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 266, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 267, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 268, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 269, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 270, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 271, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 272, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 33 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 273, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 274, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 275, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 276, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 277, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 278, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 279, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 280, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 34 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 281, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 282, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 283, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 284, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 285, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 286, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 287, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 288, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 35 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 289, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 290, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 291, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 292, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 293, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 294, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 295, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 296, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 36 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 297, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 298, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 299, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 300, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 301, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 302, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 303, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 304, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 37 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 305, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 306, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 307, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 308, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 309, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 310, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 311, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 312, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 38 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 313, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 314, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 315, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 316, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 317, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 318, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 319, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 320, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 39 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 321, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 322, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 323, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 324, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 325, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 326, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 327, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 328, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 40 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 329, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 330, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 331, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 332, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 333, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 334, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 335, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 336, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 41 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 337, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 338, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 339, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 340, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 341, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 342, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 343, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 344, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 42 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 345, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 346, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 347, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 348, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 349, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 350, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 351, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 352, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 43 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 353, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 354, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 355, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 356, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 357, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 358, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 359, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 360, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 44 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 361, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 362, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 363, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 364, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 365, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 366, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 367, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 368, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 45 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 369, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 370, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 371, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 372, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 373, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 374, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 375, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 376, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 46 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 377, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 378, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 379, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 380, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 381, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 382, ​​and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 383, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 384, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 47 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 385, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 386, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 387, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 388, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 389, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 390, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 391, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 392, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in group 1; The upstream and downstream primer sequences of the primer pairs for each site in group 48 are as follows: 1) The HLA-AF sequence is shown in SEQ ID NO. 393, and the HLA-A-R1 and HLA-A-R2 sequences are the same as those in group 1; 2) The HLA-BF sequence is shown in SEQ ID NO. 394, and the HLA-B-R1 and HLA-B-R2 sequences are the same as those in group 1; 3) The HLA-CF sequence is shown in SEQ ID NO. 395, and the HLA-C-R1 and HLA-C-R2 sequences are the same as those in group 1; 4) The HLA-DPA-R sequence is shown in SEQ ID NO. 396, and the HLA-DPA-F1 and HLA-DPA-F2 sequences are the same as those in group 1; 5) The HLA-DPB-F sequence is shown in SEQ ID NO. 397, and the HLA-DPB-R1 and HLA-DPB-R2 sequences are the same as those in group 1; 6) The HLA-DQA-F sequence is shown in SEQ ID NO. 398, and the HLA-DQA-R1 and HLA-DQA-R2 sequences are the same as those in group 1; 7) The HLA-DQB-F sequence is shown in SEQ ID NO. 399, and the HLA-DQB-R1 and HLA-DQB-R2 sequences are the same as those in group 1; 8) The HLA-DRB1345-F sequence is shown in SEQ ID NO. 400, and the HLA-DRB1345-R1 and HLA-DRB1345-R2 sequences are the same as those in Group 1.

2. A primer set for HLA ultra-high-resolution genotyping, wherein the primer set is used for HLA gene multiplex amplification, characterized in that: The primer set includes primer pairs for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3 / 4 / 5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 loci. The loci can be configured into two sets of upstream and downstream primers. The upstream and downstream primers and their related nucleotide sequences are shown in the following table:

3. Use of the primer set according to claim 1 in preparing an HLA ultra-high resolution genotyping detection kit.

4. An HLA ultra-high resolution genotyping detection kit, characterized in that: The detection kit comprises the primer set and detection reagent as claimed in claim 1.

5. The detection kit according to claim 4, characterized in that The detection reagent includes the HLA gene rapid amplification enzyme system Flash PCR Master Mix For Long Fragment.

6. Use of the primer set according to claim 2 in preparing an HLA ultra-high resolution genotyping detection kit.

7. An HLA ultra-high resolution genotyping detection kit, characterized in that: The detection kit comprises the primer set and detection reagent as claimed in claim 2.

8. The detection kit according to claim 7, characterized in that The detection reagent includes the HLA gene rapid amplification enzyme system Flash PCR Master Mix For Long Fragment and Index synthesis related oligonucleotide sequences; The Index synthesis-related oligonucleotide sequences are selected from any one or more groups of Index01-Index48. The upstream and downstream primers of the primer pairs of Index01-Index48 and the related nucleotide sequences are shown in the following table:

9. A full-length and rapid sequencing method for HLA typing based on nanopore sequencing for non-disease diagnosis and treatment purposes, characterized in that: The method uses the primer set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 8 to amplify the HLA gene in the sample to be tested, and then sequences the amplified product.

10. An HLA genotyping method for purposes other than disease diagnosis and treatment, characterized in that: The method comprises amplifying the HLA genes in the sample to be tested using the primer set according to any one of claims 1-2 or the kit according to any one of claims 3-8, constructing a sequencing library from the amplified products, and then sequencing and performing bioinformatics analysis on the sequencing data to obtain full-length ultra-high-resolution typing information including 11 HLA genes in the sample to be tested.

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