HLA (human leukocyte antigen) gene amplification primer based on third-generation sequencing platform and application of HLA gene amplification primer

Abstract

The invention provides an HLA (human leukocyte antigen) gene amplification primer based on a third-generation sequencing platform and application of the HLA gene amplification primer. The HLA gene amplification primer comprises a composition of any one or more of nine groups of primers. The amplification primer designed aiming at the 11 target spot of the HLA not only realizes near full-length amplification of the HLA gene, but also improves the amplification specificity, and further improves the homogeneity of HLA gene sequencing data. Besides, the amplification primers aiming at 11 target spots of the HLA gene are in the same reaction system, so that one-tube detection is realized, and the operation is convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid sequencing, and in particular, to an HLA gene amplification primer based on a third-generation sequencing platform and its application. Background Art

[0002] Human Leukocyte Antigen (HLA) is the expression product of the major histocompatibility complex in humans, located on the short arm of chromosome 6, and consists of a series of closely linked gene loci. The HLA system plays a crucial role in the immune system. Its main function is to encode antigen peptide-presenting molecules on the cell surface, which can present antigen peptides to the T cell receptors (TCRs) on the surface of T cells, thereby activating the immune response.

[0003] HLA is divided into class I antigens and class II antigens according to its distribution and function. HLA class I molecules are presenting molecules for endogenous antigens; HLA class II molecules are presenting molecules for exogenous antigens. The specificity of HLA class I antigens depends on the α heavy chain, which is encoded by the HLA-A, B, and C loci; its β light chain is β2-microglobulin, and the encoding gene is on chromosome 15. HLA class II antigens are controlled by the HLA-D region (including 5 subregions of nucleotide sequences), and the A gene and B gene in it encode the α heavy chain and β light chain respectively. The antigen polymorphism depends on the β light chain. Each of the above genes is a polymorphic locus (multiple alleles) and is codominant.

[0004] If the MHC is regarded as a whole, its polymorphism is even more prominent. Conservatively estimated, there are at least 1300 different haplotypes, and correspondingly about 17×10 to the seventh power of genotypes. The HLA system is one of the most polymorphic genetic systems, meaning that there are many different HLA gene variants (alleles) among different individuals. In medicine, accurate and high-precision HLA typing has important applications in aspects such as organ transplantation donor-recipient matching, autoimmune disease research, forensic kinship or identity identification, etc.

[0005] Currently, HLA detection techniques include serological typing. However, its operation is complex, prone to cross-reactions, with low precision and resolution, and it has gradually been replaced by PCR and sequencing techniques. PCR-based HLA detection techniques mainly include Sequence-Specific Oligonucleotides Probes (SSO), Sequence-Specific Primers (SSP), Sequence-Based Typing (SBT), and Next Generation Sequencing (NGS). The SSO technique determines HLA genotypes based on the hybridization signals between probes and PCR amplification products. The operation is rather cumbersome, it is difficult to detect new alleles, and it can only distinguish known typing sequences, and the interpretation of nucleotide sequences involves a certain degree of subjectivity. The SSP technique also has the problem of being unable to detect new alleles and cannot distinguish pseudogenes. The SBT technique is based on first-generation sequencing and can detect new alleles, but it cannot distinguish between two alleles and will produce ambiguous results. NGS is based on second-generation sequencing technology and can detect new alleles, but the sequencing length is generally short, and it cannot sequence the entire HLA gene. It is still limited to exons 2, 3, and 4 (loci studied more in the prior art), and still cannot obtain sequence information of introns and UTR regions. The typing precision generally reaches the 4-digit level. Due to the extremely high polymorphism of the nucleotide sequences contained in HLA, the above techniques still have problems that cannot be overcome and often obtain ambiguous results.

[0006] The prior art has used third-generation sequencing technology to perform single amplification (non-multiplex amplification) on class I HLA genes. This method only amplifies class I HLA genes (three HLA genes), with a small coverage of HLA genes, and it uses single-gene locus amplification, having the problems of cumbersome operation and high cost. Patent application CN 113817725 A utilizes the advantages of the long read length of third-generation sequencing and increases the amplification region in primer design, which can effectively provide complete HLA (including exons and introns) information and improve the sensitivity and accuracy of typing. However, this method has defects such as a large number of non-specific amplifications, poor target amplification uniformity, and high library construction costs for single samples. Therefore, it is particularly important to develop a HLA typing detection method with strong specific amplification, high target amplification uniformity, low library construction cost, and capable of effectively obtaining complete HLA information. Summary of the Invention

[0007] The main object of the present invention is to provide a HLA gene amplification primer based on a third-generation sequencing platform and its application to solve the problem of poor specificity in HLA gene amplification during HLA gene sequencing in the prior art.

[0008] To achieve the above object, according to the first aspect of the present invention, there is provided an amplification primer for HLA gene, and the amplification primer comprises a composition of any one or any plurality of the following nine groups of primers: wherein,

[0009] The first group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2;

[0010] The second group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 4;

[0011] The third group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 6;

[0012] The fourth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 8;

[0013] The fifth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 10;

[0014] The sixth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 12;

[0015] The seventh group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 14;

[0016] The eighth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 16;

[0017] The ninth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 18 and / or having the nucleotide sequence shown in SEQ ID NO: 19.

[0018] Further, among the above nine groups of primers, the molar ratio between the upstream primer and the downstream primer within each group of primers is: (0.9~1.1):(0.9~1.1).

[0019] Further, the amplification primers are a composition of any multiple groups among the above nine groups of primers; in the above composition, the molar ratio between each group of primers is: the first group of primers: the second group of primers: the third group of primers: the fourth group of primers: the fifth group of primers: the sixth group of primers: the seventh group of primers: the eighth group of primers: the ninth group of primers is (2.9 - 3.1):(1.9 - 2.1):(1.9 - 2.1):(8.9 - 9.1):(16.9 - 17.1):(10.9 - 11.1):(25.9 - 26.1):(3.9 - 4.1):(11.9 - 12.1).

[0020] Further, the 5'-end of the above amplification primers contains a tag sequence.

[0021] Further, the above tag sequence is a 6 - 10bp oligonucleotide sequence.

[0022] To achieve the above object, according to the second aspect of the present invention, a kit is provided, and the above kit includes the above amplification primers.

[0023] Further, the above kit further includes any one or more of the following reagents: PCR amplification reagents, gene purification reagents, sequencing library construction reagents or sequencing reagents; wherein, the above PCR amplification reagents include: water, dNTP, DNA polymerase and DNA polymerase buffer.

[0024] Further, the above DNA polymerase is selected from any one of the following: KOD FX Neo, KOD - Multi & Epi -, KOD FX, KOD OneTM PCR Master Mix or KOD - Plus - Neo.

[0025] To achieve the above object, according to the third aspect of the present invention, a method for HLA gene PCR amplification is provided, and the method includes: performing PCR amplification using the above amplification primers to obtain the above HLA gene amplification product.

[0026] Further, the reaction program of the above PCR amplification is selected from any one of the following:

[0027] 1) 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 4 cycles, (98°C for 10 s, 72°C for 12 min) * 4 cycles, (98°C for 10 s, 70°C for 12 min) * 4 cycles, (98°C for 10 s, 68°C for 12 min) * 18 cycles, 68°C for 7 min, store at 4°C;

[0028] 2) 94°C for 2 min, (98°C for 10 s, 72°C for 12 min) * 2 cycles, (98°C for 10 s, 70°C for 12 min) * 2 cycles, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C;

[0029] 3) 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 1 cycle, (98°C for 10 s, 73°C for 12 min) * 1 cycle, (98°C for 10 s, 72°C for 12 min) * 1 cycle, (98°C for 10 s, 71°C for 12 min) * 1 cycle, (98°C for 10 s, 70°C for 12 min) * 1 cycle, (98°C for 10 s, 69°C for 12 min) * 1 cycle, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C.

[0030] To achieve the above object, according to the fourth aspect of the present invention, there is provided a method for constructing an HLA gene sequencing library, the construction method comprising: ligating an amplification product of the HLA gene with a sequencing adapter to obtain the above HLA gene sequencing library;

[0031] The above amplification product is selected from the amplification product obtained by amplifying the above HLA gene using the above HLA gene amplification primers or the amplification product obtained by amplifying using the above HLA gene PCR amplification method.

[0032] Further, before ligating the above amplification product with the above sequencing adapter, the construction method further comprises: purifying the above amplification product.

[0033] To achieve the above object, according to the fourth aspect of the present invention, there is provided an HLA gene sequencing method, the HLA gene sequencing method comprising: sequencing a sequencing library; the above sequencing library is a sequencing library constructed by using the above HLA gene sequencing library construction method.

[0034] Further, the above sequencing uses third-generation sequencing technology.

[0035] To achieve the above object, according to the fifth aspect of the present invention, there is provided an HLA gene typing detection method, the HLA gene typing detection method comprising: detecting and typing the HLA genotype by using the above HLA gene sequencing method.

[0036] To achieve the above object, according to the sixth aspect of the present invention, there is provided an application of the above amplification primers or the above kit in the preparation of HLA gene typing detection reagents.

[0037] To achieve the above object, according to the seventh aspect of the present invention, there is provided an application of the above amplification primer, or the above kit, or the above method for HLA gene PCR amplification, or the above method for constructing an HLA gene sequencing library, or the above method for HLA gene sequencing, or the above method for HLA gene typing detection in HLA gene typing.

[0038] Further, the above HLA gene typing sequentially includes the following steps: HLA gene amplification, HLA gene sequencing library.

[0039] By applying the technical solution of the present invention, amplification primers or their compositions designed for 11 gene loci (including: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5) are used, which specifically contain the nucleotide sequences shown in SEQ ID NOs: 1-19. Using the above amplification primers or their compositions to specifically amplify and enrich the HLA gene, and mixing and sequencing the amplified fragments can effectively obtain all the information of the HLA gene (including exons and introns), realizing the near-full-length amplification of the HLA gene.

[0040] The annealing temperatures of the amplification primers designed in the present invention are similar, which improves the enrichment specificity of the HLA gene target sequence and reduces the proportion of invalid data. In addition, using the above amplification primers under the amplification program of the present invention, multiple genes can be amplified simultaneously in one tube system, and the uniformity between targets is improved, thereby improving the data utilization rate and reducing the redundancy of the subsequent obtained sequencing data. After subsequent construction of mixed libraries (library construction) and gene sequencing of multiple samples, the data uniformity between the obtained samples is good. The resolution of HLA gene sequencing data typing is improved, and 6-digit resolution of HLA typing is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0042] Figure 1 A schematic diagram of the amplification product peak map of the HLA-A gene locus according to Example 1 of the present invention is shown.

[0043] Figure 2 A schematic diagram of the amplification product peak map of the HLA-B gene locus according to Example 1 of the present invention is shown.

[0044] Figure 3Shows a schematic diagram of the amplification product peak map of the HLA-C gene locus according to Embodiment 1 of the present invention.

[0045] Figure 4 Shows a schematic diagram of the amplification product peak map of the HLA-DPA1 gene locus according to Embodiment 1 of the present invention.

[0046] Figure 5 Shows a schematic diagram of the amplification product peak map of the HLA-DPB1 gene locus according to Embodiment 1 of the present invention.

[0047] Figure 6 Shows a schematic diagram of the amplification product peak map of the HLA-DQA1 gene locus according to Embodiment 1 of the present invention.

[0048] Figure 7 Shows a schematic diagram of the amplification product peak map of the HLA-DQB1 gene locus according to Embodiment 1 of the present invention.

[0049] Figure 8 Shows a schematic diagram of the amplification product peak map of the HLA-DRB-E1 gene locus according to Embodiment 1 of the present invention.

[0050] Figure 9 Shows a schematic diagram of the amplification product peak map of the HLA-DRB-E2~6 gene loci according to Embodiment 1 of the present invention.

[0051] Figure 10 Shows a schematic diagram of the amplification product peak map of the HLA-A gene locus according to Comparative Example 1 of the present invention.

[0052] Figure 11 Shows a schematic diagram of the amplification product peak map of the HLA-B gene locus according to Comparative Example 1 of the present invention.

[0053] Figure 12 Shows a schematic diagram of the amplification product peak map of the HLA-C gene locus according to Comparative Example 1 of the present invention.

[0054] Figure 13 Shows a schematic diagram of the amplification product peak map of the HLA-DPA1 gene locus according to Comparative Example 1 of the present invention.

[0055] Figure 14 Shows a schematic diagram of the amplification product peak map of the HLA-DPB1 gene locus according to Comparative Example 1 of the present invention.

[0056] Figure 15 Shows a schematic diagram of the amplification product peak map of the HLA-DQA1 gene locus according to Comparative Example 1 of the present invention.

[0057] Figure 16Schematic diagram of the amplification product peak map of the HLA-DQB1 gene locus of Comparative Example 1 according to the present invention.

[0058] Figure 17 Schematic diagram of the amplification product peak map of the HLA-DRB-E1 gene locus of Comparative Example 1 according to the present invention.

[0059] Figure 18 Schematic diagram of the amplification product peak map of the HLA-DRB-E2E6 gene locus of Comparative Example 1 according to the present invention.

[0060] Figure 19 Schematic diagram of the average sequencing depth ratio of Example 2 and Comparative Example 2 according to the present invention.

[0061] Figure 20 Schematic diagram of the primer amplification region of the HLA gene coding locus of the present invention. Detailed implementation manners

[0062] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0063] Term explanation:

[0064] Multiplex PCR amplification: A PCR technique that simultaneously amplifies multiple target sequences in the same reaction system. It can be used to simultaneously detect multiple genes or DNA fragments, saving time and cost, and improving experimental efficiency. Multiplex PCR amplification usually requires designing multiple primers corresponding to different target sequences and optimizing the PCR reaction conditions to ensure that each target sequence can be effectively amplified. This technique is widely used in fields such as molecular biology research, disease diagnosis, and genetic analysis.

[0065] MHC: The full name is major histocompatibility complex, the human major histocompatibility complex, which is a highly polymorphic genomic region on the chromosome encoding proteins related to intercellular recognition and antigen presentation. In humans, it is called the HLA complex.

[0066] Allele: Refers to different gene forms at the same gene locus. They contain the same function but have different expression patterns. In an individual, there can be two alleles at each gene locus, one from the mother and one from the father.

[0067] HLA-DRB1 / 3 / 4 / 5: Class II genes belonging to HLA. HLA-DRB1 encodes the β-chain of the HLA-DR molecule. This β-chain forms a heterodimer with the α-chain encoded by HLA-DRA, and both are anchored in the membrane and participate in antigen presentation. HLA-DRB3 / 4 / 5 has a high homology with HLA-DRB1 and also forms a heterodimer with HLA-DRA1 for cell surface display of polypeptide antigens. However, the protein expression level of HLA-DRB3 / 4 / 5 is relatively low. For example, the expression level of HLA-DRB5 is only 20% of that of HLA-DRB1. HLA-DRB3 / 4 / 5 is adjacent to HLA-DRB1, and HLA-DRB1 is carried by all individuals and is located in the core region of the HLA-DR locus (short arm p21.3 of chromosome 6). An individual may carry 0, 1, or 2 types of HLA-DRB3 / 4 / 5 (i.e., each haplotype can carry at most one DRB3 / 4 / 5 gene), but usually does not carry multiple types of DRB3, DRB4, and DRB5 simultaneously. Moreover, which specific locus is involved has a very strong correlation with the typing of HLA-DRB1, and there is a very strong linkage disequilibrium between HLA-DRB1 and HLA-DRB3 / 4 / 5.

[0068] Gene locus: Refers to a specific position on a chromosome, corresponding to a specific gene. Different alleles can exist at each gene locus, and these alleles determine the genetic characteristics of an individual. By studying the combination and distribution of alleles at a gene locus, the genetic information and phenotypic characteristics of an individual can be understood. Gene loci play an important role in genetic research and can be used to explore genetic variation, genome-wide association, and evolution, etc.

[0069] Third-generation sequencing technology: Refers to single-molecule real-time sequencing technology. Compared with the first two generations of sequencing technologies, its greatest feature is that the sequencing process of single-molecule sequencing does not require PCR amplification, achieving the separate sequencing of each DNA molecule. Currently, the main third-generation sequencing technologies can be divided into nanopore electrical signal sequencing and single-molecule fluorescence signal sequencing according to different sequencing principles. Currently, the main third-generation sequencing technologies can be divided into: the nanopore single-molecule sequencing technology of Oxford Nanopore Technologies (ONT) and the single-molecule real-time sequencing (SMRT) technology of Pacific Biosciences (PacBio) in the United States.

[0070] As mentioned in the background art, when performing HLA gene sequencing in the prior art, there are problems of poor amplification specificity and poor sequencing uniformity. In the present invention, the inventors attempted to design specific primers with similar annealing temperatures for 11 targets of HLA and adopt gradient annealing to improve the amplification specificity and sequencing uniformity of HLA gene detection, thus proposing the protection scheme of the present invention.

[0071] In the first typical embodiment of the present invention, an amplification primer for HLA gene is provided. The amplification primer comprises a composition of any one or any combination of the following nine groups of primers: wherein,

[0072] The first group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 2;

[0073] The second group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 4;

[0074] The third group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 6;

[0075] The fourth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 8;

[0076] The fifth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 10;

[0077] The sixth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 12;

[0078] The seventh group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 14;

[0079] The eighth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 16;

[0080] The ninth group of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 18 and / or having the nucleotide sequence shown in SEQ ID NO: 19.

[0081] The HLA gene includes multiple coding sites: HLA-A (the first set of primers), HLA-B (the second set of primers), HLA-C (the third set of primers), HLA-DPA1 (the fourth set of primers), HLA-DPB1 (the fifth set of primers), HLA-DQA1 (the sixth set of primers), HLA-DQB1 (the seventh set of primers), HLA-DRB-E1 (the eighth set of primers), and HLA-DRB-E2~6 (the ninth set of primers). See Figure 20 .

[0082] It should be noted that the HLA-DRB gene family contains four gene subtypes: HLA-DRB1 / 3 / 4 / 5. Since these four gene subtypes have significant sequence similarities and all contain 6 exons, when amplifying the above four gene subtypes, the same primer set is used for amplification. That is, the eighth set of primers targets the first exon region; the ninth set of primers targets the second to sixth exon regions.

[0083] In addition, the compositions in the above "compositions of any number of sets" include mixed phases and also single phases sold or used in sets.

[0084] In the PCR amplification system, primer annealing design is one of the key influencing points affecting amplification specificity. If the designed primer annealing temperature is higher than the actual reaction annealing temperature, it will cause non-specific binding of primers to the template, resulting in non-specific amplification. If the designed primer annealing temperature is lower than the actual reaction annealing temperature, it will lead to insufficient annealing and low amplification efficiency. In a multiplex amplification system, multiple sets of primers are amplified in the same system, using the same annealing reaction temperature. Due to the differences in primer annealing temperatures among different primers, the non-specific amplification and amplification efficiency differences will be exacerbated, which makes the design of the amplification program of the multiplex system complex.

[0085] In multiplex PCR amplification, by adjusting the primer sequence length and GC content, designing a reasonable amplification primer sequence to unify the primers to a similar annealing temperature, and combining the design of a touchdown PCR amplification program, the specificity of the primer system can be enhanced while ensuring the amplification efficiency. The annealing temperatures designed for the above 9 sets of primers are close (60°C - 65°C), and the same PCR amplification conditions are uniformly used, and the specificity of the amplification primers is strong. Using the above amplification primers to amplify the HLA gene can help ensure the amplification efficiency and improve the specificity of the amplification system at the same time.

[0086] After the above nine sets of primers are mixed and subjected to multiplex PCR amplification in a single system (one tube), it can cover all the coding sites of the HLA gene. Through subsequent library construction and sequencing detection, all the information carried by the target region can be obtained, and the HLA gene typing can be accurately detected.

[0087] When multiple sets of primers are amplified in one tube, competition for amplification of substrates is generated between primers, resulting in differences in the multiples of amplification of each primer, and poor coverage uniformity of each target. Some targets may have the problem of insufficient amplification. In order to improve their coverage depth, the amount of sequencing data needs to be further increased, which increases the sequencing cost. Some targets may also have the problem of over-amplification, and too much amplification may cause sequencing data redundancy. Therefore, the difference in amplification uniformity in the same system may affect the sequencing coverage depth, thereby further affecting the uniformity of sequencing data. By adjusting the amount of each set of HLA primers in the multiple amplification, combined with the primers designed by the present invention and the corresponding PCR amplification system, the uniformity of amplification and the utilization rate of amplification data can be improved, thereby improving the coverage uniformity of sequencing data and the utilization rate of sequencing data.

[0088] The molar ratio of the upstream primer and its corresponding downstream primer in the amplification primer set may affect the amplification efficiency of the target gene. In a preferred embodiment of the present invention, in the above nine primer sets, the molar ratio between the upstream primer and the downstream primer in each primer set is: (0.9~1.1): (0.9~1.1). In a more preferred embodiment of the present invention, in the above nine primer sets, the molar ratio between the upstream primer and the downstream primer in each primer set is: 1:1. Using the above molar ratio of upstream and downstream amplification primers for PCR amplification includes the beneficial effects of high amplification efficiency and good uniformity.

[0089] It should be noted that the ninth set of primers provides two downstream primers, whose sequences are SEQ ID NO: 18 and SEQ ID NO: 19. In a preferred embodiment of the present invention, the ninth set of primers comprises an upstream primer with a nucleotide sequence of SEQ ID NO: 17 and a downstream primer with a nucleotide sequence of SEQ ID NO: 18 and / or a downstream primer with a nucleotide sequence of SEQ ID NO: 19. That is, the two downstream primers can be used simultaneously or one of them can be used at will.

[0090] In a more preferred embodiment of the present invention, the ninth set of primers comprises an upstream primer having a nucleotide sequence of SEQ ID NO: 17, a downstream primer having a nucleotide sequence of SEQ ID NO: 18, and a downstream primer having a nucleotide sequence of SEQ ID NO: 19. In a further preferred embodiment of the present invention, the molar ratio of the downstream primer having a nucleotide sequence of SEQ ID NO: 18 to the downstream primer having a nucleotide sequence of SEQ ID NO: 19 is 1:1. The use of the two downstream primers can cover different types of variation in the polymorphic region and adapt to complex gene structures. In the case of highly polymorphic or complex gene structures, targeting different specific sites has the beneficial effects of reducing nonspecific amplification, improving the accuracy, reliability and amplification efficiency of PCR results.

[0091] In a preferred embodiment of the present invention, the amplification primers are a composition of any multiple sets among nine sets of primers; in the composition, the molar ratio between each set of primers is: the first set of primers: the second set of primers: the third set of primers: the fourth set of primers: the fifth set of primers: the sixth set of primers: the seventh set of primers: the eighth set of primers: the ninth set of primers is (2.9~3.1):(1.9~2.1):(1.9~2.1):(8.9~9.1):(16.9~17.1):(10.9~11.1):(25.9~26.1):(3.9~4.1):(11.9~12.1).

[0092] Performing multiplex PCR amplification using the molar ratio of the above nine sets of primer groups can effectively improve the uniformity of coverage of each target sequence in the amplification product, thereby helping to improve the effective utilization rate of subsequent sequencing data and reducing the sequencing cost.

[0093] In a more preferred embodiment of the present invention, the molar ratio between the working concentrations of each set of primer groups is: the first set of primers: the second set of primers: the third set of primers: the fourth set of primers: the fifth set of primers: the sixth set of primers: the seventh set of primers: the eighth set of primers: the ninth set of primers = 3:2:2:9:17:11:26:4:12. Using multiple sets of amplification primers with the above molar ratio for multiplex PCR helps to improve the uniformity of amplification.

[0094] When performing HLA gene sequencing on multiple samples simultaneously, in order to improve the sequencing efficiency of the samples, in a preferred embodiment of the present invention, the 5'-end of the above amplification primers contains a tag sequence. In a more preferred embodiment of the present invention, the tag sequence is a 6- to 10-base sequence. In a further preferred embodiment of the present invention, the tag sequence is a 6- to 8-base sequence.

[0095] In a preferred embodiment of the present invention, the tag sequences satisfy that the tag sequences used for multiple samples mixed and library-constructed together or sequenced together on the same machine have the same GC content. In a more preferred embodiment of the present invention, there is no reverse complementary sequence between the above tag sequences and the primer sequences within the nine sets. The tag sequences with the same GC content can avoid the influence of sequencing preference and batch effect on the amplification efficiency and the uniformity of sequencing data, ensure the balance of sequencing signals, and further improve the accuracy of typing results.

[0096] For simultaneous sequencing of multiple samples on a machine, amplification products with different tag sequences can be directly mixed and library constructed at the library construction stage. Different samples carry different tag sequences, so that after sequencing is completed, the sequencing data can be split according to the samples through different tag information. Using amplification primers with tag sequences for amplification can improve the sequencing throughput of mixed library construction, and has the beneficial effects of convenience, high efficiency and cost reduction. The tag sequence can be any sequence combination that does not affect amplification and / or library construction operations.

[0097] In the second typical embodiment of the present invention, a kit is provided, and the kit includes the above-mentioned amplification primers. In a preferred embodiment of the present invention, the above-mentioned kit further includes any one or more of the following reagents: PCR amplification reagents, gene purification reagents, sequencing library construction reagents and sequencing reagents; wherein, the above-mentioned PCR amplification reagents include: water, dNTP, DNA polymerase and DNA polymerase buffer. Using the above-mentioned kit for HLA genotyping has the beneficial effect of simple operation. It should be noted that the above-mentioned gene purification reagents, sequencing library construction reagents and sequencing reagents are all common reagents in the art.

[0098] In a preferred embodiment of the present invention, the above-mentioned DNA polymerase is selected from any one of the following: KOD FX Neo, KOD -Multi & Epi-, KOD FX, KOD OneTM PCR Master Mix or KOD -Plus- Neo. Using the above-mentioned high-fidelity DNA polymerase can effectively improve the accuracy and consistency of the amplification products, and contribute to improving the accuracy of HLA genotyping.

[0099] In the third typical embodiment of the present invention, a method for PCR amplification of HLA genes is provided, and the method includes: performing PCR amplification using the above-mentioned amplification primers to obtain HLA gene amplification products. Using the above method to amplify HLA genes can improve the specificity and efficiency of amplification, so that the amplification of HLA genes is more accurate.

[0100] In a preferred embodiment of the present invention, the reaction program of PCR amplification is selected from any one of the following:

[0101] 1) 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 4 cycles, (98°C for 10 s, 72°C for 12 min) * 4 cycles, (98°C for 10 s, 70°C for 12 min) * 4 cycles, (98°C for 10 s, 68°C for 12 min) * 18 cycles, 68°C for 7 min, store at 4°C;

[0102] 2) 94°C for 2 min, (98°C for 10 s, 72°C for 12 min) * 2 cycles, (98°C for 10 s, 70°C for 12 min) * 2 cycles, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C;

[0103] 3) 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 1 cycle, (98°C for 10 s, 73°C for 12 min) * 1 cycle, (98°C for 10 s, 72°C for 12 min) * 1 cycle, (98°C for 10 s, 71°C for 12 min) * 1 cycle, (98°C for 10 s, 70°C for 12 min) * 1 cycle, (98°C for 10 s, 69°C for 12 min) * 1 cycle, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C. Using the above PCR amplification procedure helps to inhibit non-specific amplification, enrich the target region and improve the specificity of amplification.

[0104] In a more preferred embodiment of the present invention, the reaction procedure of the above PCR amplification is selected from the above 1) 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 4 cycles, (98°C for 10 s, 72°C for 12 min) * 4 cycles, (98°C for 10 s, 70°C for 12 min) * 4 cycles, (98°C for 10 s, 68°C for 12 min) * 18 cycles, 68°C for 7 min, store at 4°C.

[0105] In a fourth typical embodiment of the present invention, a method for constructing an HLA gene sequencing library is provided. The construction method includes: ligating the amplification product of the HLA gene with a sequencing adapter to obtain an HLA gene sequencing library; the amplification product is selected from the amplification product obtained by amplifying the above HLA gene using the above HLA gene amplification primers or the amplification product obtained by amplifying using the above HLA gene PCR amplification method. Using the above method for constructing an HLA gene sequencing library helps to improve the quality and depth of sequencing data and provides a data basis for high-resolution HLA gene typing.

[0106] In a preferred embodiment of the present invention, before ligating the above amplification product and the above sequencing adapter, the construction method further includes: purifying the above amplification product. The purpose of purification is to remove the reagents and non-target products in the PCR reaction system. In a more preferred embodiment of the present invention, the above purification method is selected from any one or more of the following: magnetic bead method or silica column method.

[0107] In the fifth typical embodiment of the present invention, a method for HLA gene sequencing is provided. The method includes sequencing a sequencing library; the sequencing library is a sequencing library constructed by using the above-mentioned method for constructing an HLA gene sequencing library. In a preferred embodiment of the present invention, the sequencing uses third-generation sequencing technology. The sequencing data obtained by using the above-mentioned sequencing method has high capture specificity and strong data uniformity, which helps to improve the accuracy of HLA gene typing, enhance the detection efficiency, and reduce the detection cost. With the characteristics of long read length and high throughput, the third-generation sequencing technology can provide more comprehensive HLA gene information, thereby helping to improve the accuracy of HLA typing.

[0108] In the sixth typical embodiment of the present invention, a method for HLA gene typing detection is provided. The HLA gene typing detection method includes: detecting and typing the HLA genotype by using the above-mentioned HLA gene sequencing method. Using this method for HLA gene typing has high accuracy and can improve the precision of HLA gene typing.

[0109] In the seventh typical embodiment of the present invention, an application of the above-mentioned amplification primer or the above-mentioned kit in the preparation of an HLA gene typing detection reagent is provided.

[0110] In the eighth typical embodiment of the present invention, an application of the above-mentioned amplification primer or the above-mentioned kit or the above-mentioned method for HLA gene PCR amplification or the above-mentioned method for constructing an HLA gene sequencing library or the above-mentioned HLA gene sequencing method or the above-mentioned HLA gene typing detection method in HLA gene typing is provided.

[0111] In a preferred embodiment of the present invention, HLA gene typing sequentially includes the following steps: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing, and HLA gene typing determination.

[0112] The following further describes the present invention in detail with specific embodiments, and these embodiments should not be construed as limiting the scope of protection required by the present invention.

[0113] Example 1: Specificity of single primer and amplification system

[0114] I. PCR amplification

[0115] For different HLA gene targets, the designed primer sequences are shown in Table 1 below:

[0116] Table 1

[0117]

[0118] In the table, F represents the forward primer, also known as the upstream primer; R represents the reverse primer, also known as the downstream primer. In addition, for HLA-DRB-E2~6, two reverse primers were designed to cover different variant types in the polymorphic region and adapt to the complex gene structure. In the case of highly polymorphic or complex gene structures, for different specific sites, the possibility of non-specific amplification is reduced, and the accuracy, reliability, and amplification efficiency of the PCR results are improved.

[0119] The PCR amplification system is shown in Table 2 below:

[0120] Table 2

[0121]

[0122] Note: NA12878 gDNA (human genomic DNA standard). It should be noted that all human genomic DNA standards of the present invention are purchased from the Coriell Institute. HLA-Prim in the table includes the upstream primer and the downstream primer. Among them, the molar ratio of the upstream and downstream primers for the same gene target is 1:1. Among them, the molar ratio of the upstream primer and two downstream primers for HLA-DRB-E2~6 is F:R1:R2 = 2:1:1.

[0123] The PCR program 1 is as follows: 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 4 cycles, (98°C for 10 s, 72°C for 12 min) * 4 cycles, (98°C for 10 s, 70°C for 12 min) * 4 cycles, (98°C for 10 s, 68°C for 12 min) * 18 cycles, 68°C for 7 min, and stored at 4°C.

[0124] II. Purification

[0125] 1. Add 1.0 volume of magnetic beads (model VAHTS DNA Clean Beads) to the reaction system, mix well, and let stand at room temperature for 8 minutes;

[0126] 2. Place it on the magnetic rack and let stand for 5 minutes until the magnetic beads are completely adsorbed, and remove the supernatant;

[0127] 3. Add 200 µL of freshly prepared 80% ethanol, let stand for 30 seconds, remove the supernatant, and repeat the washing once;

[0128] 4. Briefly centrifuge on a hand-held centrifuge to remove the residual alcohol;

[0129] 5. Resuspend with 20 µL of EB buffer and let stand at room temperature for 5 minutes;

[0130] 6. Place it on a magnetic stand and let it stand for 3 minutes, then aspirate the supernatant to obtain the amplification product.

[0131] III. Detection

[0132] The fragment distribution of the amplification product was quality inspected using an Agilent 2100 bioanalyzer.

[0133] Experimental results:

[0134] Figures 1-9 They are respectively the peak graphs of the amplification products of HLA-A(F / R), HLA-B(F / R), HLA-C(F / R), HLA-DPA1(F / R), HLA-DPB1(F / R), HLA-DQA1(F / R), HLA-DQB1(F / R), HLA-DRB-E1(F / R), and HLA-DRB-E2E6(F / R1+R2) in Example 1 (SEQ ID NOs: 1-19).

[0135] Comparative Example 1

[0136] In this comparative example, gene amplification primers in Patent Application CN 113817725 A were used for the experiment.

[0137] I. Amplification

[0138] The primer sequences are shown in Table 3 below:

[0139] Table 3

[0140]

[0141] The PCR reaction system was the same as Table 2 in Example 1;

[0142] PCR Program 4 was as follows: 94°C for 2 min, (98°C for 10 s, 68°C for 12 min <starting from the 11th cycle, each cycle increases by 30 s>) * 30 cycles, 68°C for 10 min, and store at 4°C.

[0143] II. Purification and Detection

[0144] The purification steps and the method for detecting the fragment distribution of the amplification product were the same as in Example 1.

[0145] Experimental results:

[0146] Figures 10-18Primers (SEQ ID NOs: 20 - 37) in the comparative example respectively: HLA-A (F / R), HLA-B (F / R), HLA-C (F / R), HLA-DPA1 (F / R), HLA-DPB1 (F / R), HLA-DQA1 (F / R), HLA-DQB1 (F / R), HLA-DRB-E1 (F / R) and HLA-DRB-E2E6 (F / R1 + R2) amplification product peak maps.

[0147] Analysis of experimental results:

[0148] The experimental results showed that, compared with the amplification product peak maps of the corresponding target primers in Comparative Example 1 ( Figures 10-18 ), the non-specific amplification product peak signals of the amplification product peak maps of each target primer designed in the present invention in Example 1 ( Figures 1-9 ) were lower except for the main peak, indicating that the specificity of the amplification of each target primer designed in Example 1 of the present invention was significantly improved.

[0149] Example 2: Multiplex primer amplification system and PCR program

[0150] I. Amplification

[0151] Using the primer sequences designed for different targets in Table 1, dilute with low TE buffer, and then mix according to the ratio in Table 4 below, named primer mix. Among them, the molar ratio of the upstream and downstream primers for the same gene target is: 1:1. Among them, the molar ratio of the upstream primer and the two downstream primers for HLA-DRB-E2E6 is F:R1:R2 = 2:1:1.

[0152] Table 4

[0153]

[0154] Configure the PCR amplification system according to Table 5 below:

[0155] Table 5

[0156]

[0157] The PCR reaction program is as follows:

[0158] PCR Program 1 (same as Example 1): 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 4 cycles, (98°C for 10 s, 72°C for 12 min) * 4 cycles, (98°C for 10 s, 70°C for 12 min) * 4 cycles, (98°C for 10 s, 68°C for 12 min) * 18 cycles, 68°C for 7 min, store at 4°C.

[0159] PCR Program 2: 94°C for 2 min, (98°C for 10 s, 72°C for 12 min) * 2 cycles, (98°C for 10 s, 70°C for 12 min) * 2 cycles, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C.

[0160] PCR Program 3: 94°C for 2 min, (98°C for 10 s, 74°C for 12 min) * 1 cycle, (98°C for 10 s, 73°C for 12 min) * 1 cycle, (98°C for 10 s, 72°C for 12 min) * 1 cycle, (98°C for 10 s, 71°C for 12 min) * 1 cycle, (98°C for 10 s, 70°C for 12 min) * 1 cycle, (98°C for 10 s, 69°C for 12 min) * 1 cycle, (98°C for 10 s, 68°C for 12 min) * 21 cycles, 68°C for 7 min, store at 4°C.

[0161] PCR Program 4 (same as Comparative Example 1): 94°C for 2 min, (98°C for 10 s, 68°C for 12 min <starting from the 11th cycle, increasing by 30 s per cycle>) * 30 cycles, 68°C for 10 min, store at 4°C.

[0162] II. Purification

[0163] The purification steps are the same as those in Example 1.

[0164] III. Library Construction and Sequencing

[0165] The library was constructed according to the library construction process of the PacBio SMRTbell prep kit 3.0. The library was quality inspected for fragment distribution using the Agilent Femto P µLse system. The on-machine sequencing was performed according to the on-machine process and reagents recommended by PacBio Revio.

[0166] Comparative Example 2: Multiplex Primer Amplification

[0167] In this comparative example, the gene amplification primers in Patent Application CN 113817725 A were used for the experiment. The designed primer sequences were the same as those in Table 3 of Comparative Example 1.

[0168] The primers in Table 3 were mixed according to the following Table 6 (named total primer). Among them, the molar ratio of the upstream and downstream primers for the same gene target was 1:1. The molar ratio of the upstream primer and the two downstream primers for HLA-DRB-E2~6 was F:R1:R2 = 2:1:1.

[0169] Table 6

[0170]

[0171] The PCR reaction system is shown in Table 7 below:

[0172] Table 7

[0173]

[0174] The PCR reaction procedure is the same as that of Comparative Example 1.

[0175] II. Purification

[0176] The purification steps are the same as those in Example 1.

[0177] III. Library construction and sequencing

[0178] The library construction and sequencing steps are the same as those in Example 2.

[0179] Experimental results: Refer to Table 8, Table 9 and Figure 19 .

[0180] Table 8

[0181]

[0182] Table 9:

[0183]

[0184] It should be noted that the capture specificity refers to the proportion of the effective data volume aligned to the target region in the total data volume.

[0185] Analysis of experimental results:

[0186] 1) From the perspective of capture specificity

[0187] The results of Example 2 are shown in Table 8, Table 9 and Figure 19 As shown, in the same system, all 11 sites can be normally detected. Compared with Comparative Example 2, the capture specificity of the group using the amplification primers of the present invention is higher. For example, Example 2 - PCR Program 1, Example 2 - PCR Program 2, Example 2 - PCR Program 3 and Example 2 - PCR Program 4. It shows that the primers designed by the present invention have better capture specificity in multiplex amplification within the same system.

[0188] In addition, compared with the PCR programs without gradient annealing (Example 2 - PCR Program 4 and Comparative Example 2), the PCR program with a gradient annealing step from high to low has higher capture specificity for amplification, indicating that gradient annealing has an obvious promoting effect on amplification specificity. For example, Example 2 - PCR Program 1, Example 2 - PCR Program 2 and Example 2 - PCR Program 3.

[0189] Generally speaking, compared with Comparative Example 2, the embodiments 2 - PCR programs 1 to 4 designed in the present invention have all improved in target capture specificity, with the lowest specificity increase being 4 percentage points and the highest being 8 percentage points.

[0190] 2) From the perspective of the uniformity of sequencing depth

[0191] Compared with Comparative Example 2, the CV values (coefficient of variation) of the sequencing depths of each target in the embodiments 2 - PCR programs 1 - 4 of the present invention are lower, indicating that the output uniformity of the present invention is better. Among them, the CV value of the embodiment 2 - PCR program 1 is the lowest and the uniformity is the best.

[0192] For genotyping identification, it is required that each target needs to meet a certain coverage depth, that is, the target with the lowest average depth ratio determines the lower limit of data utilization. Taking the data corresponding to the HLA - DRB - E2~E6 exons with the lowest average sequencing depth as an example: in the embodiment 2 - PCR program 1, when the proportion of the lowest average depth of HLA - DRB - E2~E6 is 5.1%, and the required lowest sequencing depth is 100×, the total data volume depth to be measured is 1960× (the calculation formula is: 100× / 5.1% = 1960×).

[0193] Similarly, when calculating the proportion of 0.2% of the lowest average depth of HLA - DRB - E2E6 in Comparative Example 2, to meet the 100× depth of this target, the data volume depth to be measured is 50000× (the calculation formula is: 100× / 0.2% = 50000×). It can be seen that the improved scheme of the embodiment 2 - PCR program 1 of the present invention only requires 3.9% of the data volume of Comparative Example 2 (the calculation formula is: 1960× / 50000× = 3.9%), greatly saving the sequencing cost.

[0194] Example 3: Multiplex amplification system containing barcode primers and genotyping detection

[0195] For different targets, the designed primer sequences are as shown in Table 10 below (where the underlined NNNNNNNN represents the barcode sequence, and the rest are primer sequences):

[0196] Table 10

[0197]

[0198] The barcode sequence used in this experiment is:

[0199] Table 11

[0200]

[0201] The primers were diluted with low TE buffer and then mixed according to the ratio in Example 2, named primermix;

[0202] Configure the PCR amplification system according to Example 2; the PCR reaction program is the same as that in Example 1.

[0203] II. Purification

[0204] The purification steps are the same as those in Example 1.

[0205] III. Library construction and sequencing

[0206] The above 6 groups of PCR products were mixed in equal mass, and library construction was carried out according to the library construction process of the pacbio SMRTbell prep kit 3.0. The library was quality inspected for fragment distribution using the Agilent Femto Pulse system. The on-machine sequencing was operated according to the on-machine process recommended by PacBio Revio.

[0207] IV. Compare the sequencing data with the data recorded in the IMGT HLA database to analyze and determine the specific typing results.

[0208] Experimental results:

[0209] 6 samples were amplified and mixed for library construction using 6 barcode primers respectively. The split data volumes are shown in the following table. The output data volume ratios are basically the same, close to the theoretical ratio of 16%, indicating that the splitting is normal after amplification and mixed library construction using barcode primers.

[0210] Table 12

[0211]

[0212] As shown in Table 12, from the results of the mixed library construction and sequencing data splitting of the 6 samples obtained by using the scheme designed by the present invention, it can be seen that the present invention has good library construction sequencing data yield and uniformity. Using the above sequencing data to further type the samples, the typing results are shown in Table 13-1 and Table 13-2.

[0213] Table 13-1

[0214]

[0215] Table 13-2

[0216]

[0217] (For reference typing, see: Bettinotti P M, Ferriola D, Duke L J, et al. Characterization of 108 Genomic DNA Reference Materials for 11 Human Leukocyte Antigen (HLA) Loci: A GeT-RM Collaborative Project [J]. The Journal of Molecular Diagnostics, 2018, 20 (5): 703-715.)

[0218] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0219] The present invention designs primers for 11 targets of HLA to achieve near-full-length amplification of HLA genes. The amplified products are sequenced using a third-generation sequencing platform to obtain the full-length sequences of HLA genes, improving the resolution of HLA typing, enabling 6-digit typing with 100% accuracy, and the 11-target amplification primers are in the same reaction system to achieve one-tube detection.

[0220] In terms of primer design, the present invention adjusts the sequence length of the amplification primers to unify the annealing temperature of the amplification primers, which helps to improve the specificity of amplification. In addition, the PCR amplification program of the present invention uses gradient annealing to amplify from high to low annealing temperature, which can effectively inhibit non-specific amplification, enrich the amplification of the target region, and improve the specificity of amplification.

[0221] The present invention can improve the amplification uniformity of primers by adjusting the usage ratio between primers, thereby improving the data utilization rate.

[0222] Barcodes are added to the primer ends, and different samples are amplified using different barcodes. After amplification of multiple samples, it is possible to construct a mixed third-generation library for multiple samples, thereby achieving the sharing of the third-generation library construction cost, reducing the third-generation library construction cost and increasing the library construction throughput. At the same time, the system design of this solution ensures the data output of the mixed library construction and the uniformity of data splitting for different samples.

[0223] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A primer for amplifying HLA gene, characterized in that: The amplification primers include any one of the following nine groups of primers or a combination of any multiple groups: wherein, The first set of primers includes an upstream primer having a nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 2; The second set of primers includes an upstream primer having a nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 4; The third set of primers comprises an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 6; The fourth set of primers includes an upstream primer having a nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 8; The fifth set of primers comprises an upstream primer having a nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 10; The sixth set of primers includes an upstream primer having a nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 12; The seventh set of primers includes an upstream primer having a nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 14; The eighth set of primers comprises an upstream primer having a nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer having a nucleotide sequence shown in SEQ ID NO: 16; The ninth set of primers comprises an upstream primer having the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer having the nucleotide sequence shown in SEQ ID NO: 18 and / or having the nucleotide sequence shown in SEQ ID NO:

19.

2. The amplification primer according to claim 1, characterized in that In the nine sets of primers, the molar ratio between the upstream primer and the downstream primer in each set of primers is: (0.9~1.1): (0.9~1.1).

3. The amplification primer according to claim 2, characterized in that The amplification primers are a combination of any multiple groups of the nine groups of primers; in the combination, the molar ratio between the primer groups is: The first group of primers: the second group of primers: the third group of primers: the fourth group of primers: the fifth group of primers: the sixth group of primers: the seventh group of primers: the eighth group of primers: the ninth group of primers are (2.9~3.1): (1.9~2.1): (1.9~2.1): (8.9~9.1): (16.9~17.1): (10.9~11.1): (25.9~26.1): (3.9~4.1): (11.9~12.1).

4. The amplification primer according to claim 1, characterized in that The 5' end of the amplification primer contains a tag sequence.

5. The amplification primer according to claim 4, characterized in that The tag sequence is a 6-10 bp oligonucleotide sequence.

6. A kit, characterized in that: The kit comprises the amplification primers according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit further comprises any one or more of the following reagents: PCR amplification reagent, gene purification reagent, sequencing library construction reagent or sequencing reagent; wherein the PCR amplification reagent comprises: water, dNTP, DNA polymerase and DNA polymerase buffer.

8. The kit according to claim 7, characterized in that The DNA polymerase is selected from any one of the following: KODFX Neo, KOD -Multi & Epi-, KOD FX, KOD OneTM PCR Master Mix or KOD -Plus- Neo.

9. A method for PCR amplification of HLA genes, characterized in that: The method comprises: performing PCR amplification using the amplification primers described in any one of claims 1 to 5 to obtain the HLA gene amplification product.

10. The method according to claim 9, characterized in that The reaction procedure of the PCR amplification is selected from any one of the following: 1) 94℃ 2min, (98℃ 10s, 74℃ 12min)*4 cycles, (98℃ 10s, 72℃ 12min)*4 cycles, (98℃ 10s, 70℃ 12min)*4 cycles, (98℃ 10s, 68℃ 12min)*18 cycles, 68℃ 7min, store at 4℃; 2) 94℃ 2min, (98℃ 10s, 72℃ 12min)*2 cycles, (98℃ 10s, 70℃ 12min)*2 cycles, (98℃ 10s, 68℃ 12min)*21 cycles, 68℃ 7min, store at 4℃; 3) 94℃ 2min, (98℃ 10s, 74℃ 12min)*1 cycle, (98℃ 10s, 73℃ 12min)*1 cycle, (98℃ 10s, 72℃ 12min)*1 cycle, (98℃ 10s, 71℃ 12min)*1 cycle, (98℃ 10s, 70℃ 12min)*1 cycle, (98℃ 10s, 69℃ 12min)*1 cycle, (98℃ 10s, 68℃ 12min)*21 cycles, 68℃ 7min, store at 4℃.

11. A method for constructing an HLA gene sequencing library, characterized in that: The construction method comprises: connecting the amplified product of the HLA gene with a sequencing adapter to obtain the HLA gene sequencing library; The amplified product is selected from the amplified product obtained by amplifying the HLA gene using the HLA gene amplification primers described in any one of claims 1 to 5 or the amplified product obtained by amplifying the HLA gene using the HLA gene PCR amplification method described in claim 9 or 10.

12. The construction method according to claim 11, characterized in that: Before connecting the amplified product to the sequencing adapter, the construction method further comprises: purifying the amplified product.

13. A method for HLA gene sequencing, characterized in that: The HLA gene sequencing method comprises: sequencing a sequencing library; the sequencing library is a sequencing library constructed using the HLA gene sequencing library construction method according to claim 11 or 12.

14. The sequencing method according to claim 13, characterized in that: The sequencing adopts the third generation sequencing technology.

15. A method for detecting HLA genotyping, characterized in that: The HLA genotyping detection method comprises: using the HLA gene sequencing method according to claim 13 or 14 to detect and type the HLA genotype.

16. Use of the amplification primer according to any one of claims 1 to 5 or the kit according to any one of claims 6 to 8 in preparing an HLA genotyping detection reagent.

17. Use of the amplification primers according to any one of claims 1 to 5, the kit according to any one of claims 6 to 8, the method for PCR amplification of HLA genes according to any one of claims 9 to 10, the method for constructing an HLA gene sequencing library according to claim 11 or 12, the HLA gene sequencing method according to claim 13 or 14, or the HLA genotyping detection method according to claim 15 in HLA genotyping.

18. The use according to claim 17, characterized in that The HLA genotyping includes the following steps in sequence: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing and HLA genotyping determination.

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