PCR amplification method of HLA gene and application of PCR amplification method

Abstract

The invention provides a PCR (polymerase chain reaction) amplification method of an HLA (human leukocyte antigen) gene and application thereof. According to the HLA gene amplification method, an optimized gradient cooling PCT amplification program is adopted, and an amplification primer designed aiming at 11 target spots of HLA is combined, so that near full-length amplification of the HLA gene is realized, the amplification specificity is also improved, and the uniformity of HLA gene sequencing data is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid sequencing, and in particular, to a PCR amplification method for HLA genes and its applications. 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] HLAs are classified into class I antigens and class II antigens according to their distribution and functions. 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 with 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 more prominent. Conservatively estimated, there are at least 1300 different haplotypes, and correspondingly about 17×10^7 genotypes. The HLA system is one of the most polymorphic genetic systems, which means that there are many different HLA gene variants (alleles) among different individuals. In medicine, accurate and high-precision HLA typing matching has important applications in organ transplantation donor-recipient matching, autoimmune disease research, forensic kinship or identity identification, etc.

[0005] Currently, HLA detection techniques include serological typing, but its operation is complex, prone to cross-reactions, and has low accuracy and resolution. Currently, it has been gradually replaced by PCR and sequencing techniques. The HLA detection techniques based on PCR mainly include Sequence-Specific Oligonucleotides Probes;

[0006] Sequence-Specific Oligonucleotide (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 of probes and PCR amplification products. The operation is relatively cumbersome, it is difficult to detect new alleles, it can only distinguish known typing sequences, and the interpretation of nucleotide sequences involves a certain degree of subjectivity. The SSP 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 accuracy generally reaches the 4-digit level. Due to the extremely high polymorphism of the nucleotide sequences contained in HLA, the above techniques still have insurmountable problems and often result in ambiguous results.

[0007] 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. Moreover, this method uses single-gene locus amplification, which has the problems of cumbersome operation and high cost. Patent application CN

[0008] 113817725A takes advantage 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 high non-specific amplification, poor target amplification uniformity, and high library construction cost for single samples. Therefore, it is particularly important to develop a detection method for HLA typing with strong specific amplification, high target amplification uniformity, low library construction cost, and the ability to effectively obtain complete HLA information. Summary of the Invention

[0009] The main objective of the present invention is to provide a PCR amplification method for HLA genes and its application to solve the problem of poor specificity in HLA gene amplification during HLA gene sequencing in the prior art.

[0010] To achieve the above objective, according to the first aspect of the present invention, there is provided a PCR amplification method for HLA genes, characterized in that the method includes amplifying HLA genes using the PCR amplification program shown by the following formula; the formula is:

[0011]

[0012] Among them, in the above annealing cycle, the temperature T of the annealing extension n from T start to T end gradually decreases, and the temperature gradient of the decrease is ΔT. N stage cycles are carried out at each annealing temperature; in the above-mentioned end preservation, it is continuously kept at a constant temperature at T 恒温 temperature;

[0013] The above T n = T start - n·ΔT (n = 0, 1, 2,...), where T start ≥ T n ≥ T end ;

[0014] The above T start is 72°C to 74°C; the above T end is 66°C to 70°C; the above ΔT is 0.5°C to 2°C;

[0015] The above H is 10 min to 18 min;

[0016] The above Among them, N 总 is 25 to 35 cycles;

[0017] The above N stage is 1 to 6 cycles;

[0018] The above T0 is 94°C to 98°C; the above H0 is 30 s to 5 min; the above T1 is 94°C to 98°C; the above H1 is 10 s to 1 min; the above H2 is 5 min to 10 min.

[0019] Furthermore, the above T end is 68°C;

[0020] Optionally, the H is 10 min to 15 min;

[0021] Optionally, the N 总 is 25 to 30 cycles;

[0022] Optionally, the N stage is 1 to 4 cycles;

[0023] Optionally, the ΔT is 1°C to 2°C;

[0024] Optionally, the H is 12 min;

[0025] Optionally, the T0 is 94°C;

[0026] Optionally, the H0 is 2 min to 5 min;

[0027] Optionally, the H0 is 2 min;

[0028] Optionally, the T1 is 98 °C;

[0029] Optionally, the H1 is 10 s;

[0030] Optionally, the H2 is 7 min;

[0031] Optionally, the T 恒温 is 4 °C to 25 °C

[0032] Optionally, the T 恒温 is 4 °C.

[0033] Furthermore, the above method includes: the reaction program of the PCR amplification is selected from any one of the following:

[0034] 1) 94 °C × 2 min, (98 °C × 10 s, 74 °C × 12 min) × 4 cycles, (98 °C × 10 s, 72 °C × 12 min) × 4 cycles, (98 °C × 10 s, 70 °C × 12 min) × 4 cycles, (98 °C × 10 s, 68 °C × 12 min) × 18 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation;

[0035] 2) 94 °C × 2 min, (98 °C × 10 s, 72 °C × 12 min) × 2 cycles, (98 °C × 10 s, 70 °C × 12 min) × 2 cycles, (98 °C × 10 s, 68 °C × 12 min) × 21 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation;

[0036] 3) 94 °C × 2 min, (98 °C × 10 s, 74 °C 12 min) × 1 cycle, (98 °C × 10 s, 73 °C × 12 min) × 1 cycle, (98 °C × 10 s, 72 °C × 12 min) × 1 cycle, (98 °C × 10 s, 71 °C × 12 min) × 1 cycle, (98 °C × 10 s, 70 °C × 12 min) × 1 cycle, (98 °C × 10 s, 69 °C × 12 min) × 1 cycle, (98 °C × 10 s, 68 °C × 12 min) × 21 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation.

[0037] Furthermore, the above method uses a composition containing any one or any combination of the following nine groups of primers: wherein,

[0038] The first set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 1 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 2;

[0039] The second set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 4;

[0040] The third set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 6;

[0041] The fourth set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 7 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 8;

[0042] The fifth set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 9 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 10;

[0043] The sixth set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 11 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 12;

[0044] The seventh set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 13 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 14;

[0045] The eighth set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 15 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 16;

[0046] The ninth set of primers includes an upstream primer with the nucleotide sequence shown in SEQ ID NO: 17 and a downstream primer with the nucleotide sequence shown in SEQ ID NO: 18 and / or with the nucleotide sequence shown in SEQ ID NO: 19.

[0047] Furthermore, among the above nine sets of primers, the molar ratio between the upstream primer and the downstream primer within each set of primers is:

[0048] (0.9~1.1):(0.9~1.1).

[0049] 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):

[0050] (16.9 - 17.1):(10.9 - 11.1):(25.9 - 26.1):(3.9 - 4.1):(11.9 - 12.1).

[0051] Further, the 5' end of the above amplification primers contains a tag sequence.

[0052] Further, the above tag sequence is a 6 - 10bp oligonucleotide sequence.

[0053] To achieve the above object, according to the second aspect of the present invention, a method for constructing an HLA gene sequencing library, the above construction method includes: ligating the amplification product of the HLA gene with a sequencing adapter to obtain the HLA gene sequencing library;

[0054] The above amplification product is an amplification product obtained by amplifying using the above PCR amplification method of the HLA gene.

[0055] Further, before ligating the above amplification product with the above sequencing adapter, the above construction method further includes: purifying the above amplification product.

[0056] To achieve the above object, according to the third aspect of the present invention, a method for sequencing an HLA gene is provided, the HLA gene sequencing method includes: sequencing a sequencing library; the above sequencing library is a sequencing library constructed by using the above method for constructing an HLA gene sequencing library.

[0057] Further, the above sequencing uses third - generation sequencing technology.

[0058] To achieve the above object, according to the fourth aspect of the present invention, a method for detecting HLA gene typing is provided, the HLA gene typing detection method includes: detecting and typing the HLA genotype by using the above HLA gene sequencing method.

[0059] To achieve the above object, according to the fifth aspect of the present invention, an application of the above method for PCR amplification of the HLA gene or the above method for constructing an HLA gene sequencing library or the above method for sequencing an HLA gene or the above method for detecting HLA gene typing in HLA gene typing detection is provided.

[0060] Furthermore, the above HLA genotyping detection sequentially includes the following steps: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing, and HLA gene typing determination.

[0061] Applying the technical solution of the present invention, an optimized touchdown annealing PCR program is adopted, and a two-step PCR scheme is used to combine the annealing step and the extension step into one step at the same temperature. The annealing temperature range, annealing temperature gradient, and the ratio of amplification cycles to stabilization cycles are optimized. At the same time, 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, specifically including the nucleotide sequences shown in SEQ ID NOs: 1-19. Specific amplification and enrichment of HLA genes are carried out, and the amplified fragments are mixed and library-constructed and sequenced, which can effectively obtain all the information of HLA genes (including exons and introns), and achieve nearly full-length amplification of HLA genes.

[0062] The annealing temperatures of the amplification primers designed by the present invention are similar, and the PCR amplification program is optimized, which improves the enrichment specificity of HLA gene target sequences 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 samples is good. The resolution of HLA gene sequencing data typing is improved, and 6-digit resolution of HLA typing is achieved. Description of the Drawings

[0063] The specification drawings forming 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:

[0064] Figure 1 It is a schematic diagram of the HLA gene amplification program in Example 1 and Example 2 of the present invention.

[0065] Figure 2 It is a schematic diagram of the amplification product peak map of each gene locus in Example 1 of the present invention.

[0066] Figure 3 It is a schematic diagram of the amplification product peak map of each gene locus in Comparative Example 1 of the present invention.

[0067] Figure 4 Schematic diagram of the primer amplification region of the HLA gene coding locus of the present invention.

[0068] Figure 5 Schematic diagram of the average sequencing depth ratio according to Example 2 and Comparative Example 2 of the present invention.

[0069] Among them, T n : In the PCR amplification program, the annealing temperature set in the annealing cycle program, which decreases gradually from T start to T end , with a temperature decrease gradient of ΔT, and it decreases by ΔT degrees Celsius (°C) every N stage cycles; T start : In the PCR amplification program, the starting annealing temperature set in the annealing cycle program; T end : In the PCR amplification program, the final annealing extension temperature set in the annealing cycle program; ΔT: The temperature decrease per stage of the annealing extension temperature set in the PCR amplification program; N stage : In the PCR amplification program, the number of cycles in each annealing extension temperature stage set in the annealing cycle program; N final : In the PCR amplification program, the number of cycles set in the stable cycle program; N 总 : In the PCR amplification program, the total number of cycles in the annealing cycle program and the stable cycle program; T0: The pre-denaturation temperature set in the pre-denaturation program of the PCR amplification program; H0: The duration of the pre-denaturation temperature set in the pre-denaturation program of the PCR amplification program; T1: The denaturation temperature set in the annealing cycle program and the stable cycle program of the PCR amplification program; H1: The duration of the denaturation temperature set in the annealing cycle program and the stable cycle program of the PCR amplification program; H: The duration of the annealing extension temperature set in the annealing cycle program and the stable cycle program of the PCR amplification program; H2: The duration of the extension temperature set in the final extension program of the PCR amplification program, T 恒温 : In the PCR amplification program, the constant temperature storage temperature set in the end storage program. Detailed implementation manners

[0070] 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.

[0071] Term explanation:

[0072] Multiplex PCR amplification: A PCR technique that simultaneously amplifies multiple target sequences in the same reaction system. It can be used to detect multiple genes or DNA fragments simultaneously, 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.

[0073] MHC: The full name is major histocompatibility complex, the major histocompatibility complex in humans, 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.

[0074] Allele: Refers to different gene forms at the same locus. They have the same function but different expression patterns. In an individual, there can be two alleles at each locus, one from the mother and one from the father.

[0075] HLA-DRB1 / 3 / 4 / 5: Belong to the class II genes of 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. The positions of HLA-DRB3 / 4 / 5 are adjacent to HLA-DRB1. 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). While individuals may carry 0, 1, or 2 types of HLA-DRB3 / 4 / 5 (that is, each haplotype can carry at most one DRB3 / 4 / 5 gene), but usually do not carry multiple types of DRB3, DRB4, and DRB5 simultaneously. And specifically which locus it is 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.

[0076] Gene locus: Refers to a specific position on the 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 the gene locus, the genetic information and phenotypic characteristics of an individual can be understood. Gene locus plays an important role in genetic research and can be used to explore genetic variation, genome-wide association, and evolution and other issues.

[0077] Third-generation sequencing technology: Refers to single-molecule real-time sequencing technology. Compared with the first two generations of sequencing technologies, its biggest feature is that the sequencing process of single-molecule sequencing does not require PCR amplification, realizing 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 (Single-molecule Nanopore DNA Sequencing) technology of Oxford Nanopore Technologies (ONT) and the single-molecule real-time sequencing (Single Molecule Realtime, SMRT) technology of Pacific Biosciences (PacBio) in the United States.

[0078] As mentioned in the background technology, when sequencing HLA genes in the prior art, there are problems of poor amplification specificity and poor sequencing uniformity. In the present invention, the inventor attempts 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.

[0079] In the first typical embodiment of the present invention, a PCR amplification method for HLA genes is provided, which includes amplifying HLA genes using the PCR amplification program shown by the following formula; the formula is:

[0080]

[0081] where, T n : In the PCR amplification program, the annealing temperature set in the annealing cycle program, which decreases gradually from T start to T end , the cooling gradient is ΔT, and it decreases by ΔT degrees Celsius (°C) every N stage cycles; T start : In the PCR amplification program, the starting annealing temperature set in the annealing cycle program; T end : In the PCR amplification program, the final annealing extension temperature set in the annealing cycle program; ΔT: In the PCR amplification program, the temperature drop of each stage of annealing extension set in the annealing cycle program; N stage : In the PCR amplification program, the number of cycles set in each stage of annealing extension temperature in the annealing cycle program; N final : In the PCR amplification program, the number of cycles set in the stable cycle program; N 总: In the PCR amplification program, the total number of cycles in the annealing cycle program and the stabilization cycle program; T0: In the PCR amplification program, the pre-denaturation temperature set in the pre-denaturation program; H0: In the PCR amplification program, the duration of the pre-denaturation temperature set in the pre-denaturation program; T1: In the PCR amplification program, the denaturation temperature set in the annealing cycle program and the stabilization cycle program; H1: In the PCR amplification program, the duration of the denaturation temperature set in the annealing cycle program and the stabilization cycle program; H: In the PCR amplification program, the duration during which the annealing extension temperature set in the annealing cycle program and the stabilization cycle program lasts; H2: In the PCR amplification program, the duration of the extension temperature set in the final extension program, T 恒温 : In the PCR amplification program, the constant temperature storage temperature set in the end storage program.

[0082] In a specific embodiment, the PCR program includes: pre-denaturation, annealing cycle, stabilization cycle, final extension, and end storage.

[0083] Among them, the pre-denaturation program is set to be at temperature T0 for a duration of H0. In this step, the template DNA in the amplification system is denatured into single strands in this program, providing a basis for primer binding.

[0084] The annealing cycle program includes a cycle unit composed of two steps: denaturation and annealing extension. Among them, the denaturation step is set to be at temperature T1 for a duration of H1; the annealing extension step is set to be at temperature Tn for a duration of H. Specifically, the temperature T n decreases from T startt to T end in a gradient manner, with a temperature gradient of ΔT, and it decreases by ΔT degrees Celsius (°C) every N stage cycles. Specifically, T n = T start - n·ΔT, (n =

[0085] 0, 1, 2,...), where T start ≥ T n ≥ T end . In the annealing cycle program, the number of cycles of the cycle unit composed of the denaturation and annealing extension steps corresponding to each annealing temperature T n is N stage cycles. In this step, the target product in the amplification system occupies the template advantage in the early cycles, increasing the amplification specificity.

[0086] The stabilization cycle program includes a cycle unit composed of two steps: denaturation and annealing extension. Among them, the denaturation step is set to be at temperature T1 for a duration of H1; the annealing extension step is set to be at temperature T end for a duration of H. In the stabilization cycle program, the number of cycles of the cycle unit composed of the denaturation and annealing extension steps is Nfinal cycles. In this step, in the amplification system, at the optimal amplification temperature, the primers stably bind to the template DNA to ensure exponential accumulation of the target product. At the same time, on the basis of the high-specificity cycling enrichment of the target fragment in the previous stage, the specific advantages are accumulated while balancing the yield, amplification fidelity and amplification uniformity of each primer.

[0087] The final extension program is set to last for H2 duration at temperature T end In this step, the incomplete products in the amplification system are supplemented to ensure that all amplified fragments complete the 3'-end extension and repair breaks or single-stranded overhangs at high temperature, ensuring the amplification efficiency.

[0088] The ending preservation program is set to be kept at a constant temperature at temperature T 恒温 for a certain period of time.

[0089] It should be noted that amplifying the HLA gene by the above method can improve the specificity, efficiency and uniformity of amplification, thus making the amplification of the HLA gene more accurate.

[0090] Specifically, wherein, N 总 is 25 to 35 cycles; preferably, N 总 is 25 to 30 cycles.

[0091] In a specific embodiment, the above N stage is 1 to 6 cycles; preferably, N stage is 1 to 4 cycles.

[0092] In a specific embodiment, the above T start is 72°C to 74°C; T end is 66°C to 70°C; ΔT is 0.5°C to 2°C; H is 10 min to 18 min; preferably, the above T end is 68°C. Preferably, the above ΔT is 1°C

[0093] to 2°C. Preferably, the above H is 10 min to 15 min; more preferably, the above H is 12 min.

[0094] In a specific embodiment, the above T0 is 94°C to 98°C; preferably, the above T0 is 94°C.

[0095] In a specific embodiment, the above H0 is 30 s to 5 min; preferably, the above H0 is 2 min to 5 min; more preferably, the above H0 is 2 min.

[0096] In a specific embodiment, the above T1 is 94°C to 98°C; preferably, the above T1 is 98°C.

[0097] In a specific embodiment, the above H1 is 10 s to 1 min; preferably, the above H1 is 10 s.

[0098] In a specific embodiment, the above H2 is 5 min to 10 min; preferably, the above H2 is 7 min.

[0099] In a specific embodiment, the above T 恒温 is 4 °C to 25 °C; preferably, the above T 恒温 is 4 °C.

[0100] In a specific embodiment, the reaction program of the above-mentioned PCR amplification is selected from any one of the following:

[0101] 1) 94 °C × 2 min, (98 °C × 10 s, 74 °C × 12 min) × 4 cycles, (98 °C × 10 s, 72 °C × 12 min) × 4 cycles, (98 °C × 10 s, 70 °C × 12 min) × 4 cycles, (98 °C × 10 s, 68 °C × 12 min) × 18 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation;

[0102] 2) 94 °C × 2 min, (98 °C × 10 s, 72 °C × 12 min) × 2 cycles, (98 °C × 10 s, 70 °C × 12 min) × 2 cycles, (98 °C × 10 s, 68 °C × 12 min) × 21 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation;

[0103] 3) 94 °C × 2 min, (98 °C × 10 s, 74 °C 12 min) × 1 cycle, (98 °C × 10 s, 73 °C × 12 min) × 1 cycle, (98 °C × 10 s, 72 °C × 12 min) × 1 cycle, (98 °C × 10 s, 71 °C × 12 min) × 1 cycle, (98 °C × 10 s, 70 °C × 12 min) × 1 cycle, (98 °C × 10 s, 69 °C × 12 min) × 1 cycle, (98 °C × 10 s, 68 °C × 12 min) × 21 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation.

[0104] In a more preferred embodiment of the present invention, the reaction program of the above-mentioned PCR amplification is selected from the above 1) 94 °C × 2 min, (98 °C × 10 s, 74 °C × 12 min) × 4 cycles, (98 °C × 10 s, 72 °C × 12 min) × 4 cycles, (98 °C × 10 s, 70 °C × 12 min) × 4 cycles, (98 °C × 10 s, 68 °C × 12 min) × 18 cycles, 68 °C × 7 min, 4 °C × constant temperature preservation.

[0105] In a specific embodiment, the above amplification method uses a composition comprising any one or any combination of the following nine sets of primers: wherein,

[0106] The first set 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;

[0107] The second set 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;

[0108] The third set 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;

[0109] The fourth set 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;

[0110] The fifth set 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;

[0111] The sixth set 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;

[0112] The seventh set 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;

[0113] The eighth set 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;

[0114] 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.

[0115] 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 specifically Figure 4 .

[0116] 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 similarity 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.

[0117] In addition, the compositions in the above "compositions of any multiple sets" include mixed phases and also single phases sold or used in sets.

[0118] In the PCR amplification system, the 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 the primer 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. The difference in primer annealing temperature among different primers will exacerbate the non-specific amplification and amplification efficiency differences, which makes the design of the amplification program of the multiplex system complex.

[0119] 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 with the design of a touchdown PCR amplification program, it is possible to enhance the specificity of the primer system 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 while improving the specificity of the amplification system.

[0120] 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.

[0121] 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.

[0122] 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. The use of the upstream and downstream amplification primers of the above molar ratio for PCR amplification includes the beneficial effects of high amplification efficiency and good uniformity.

[0123] 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.

[0124] 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.

[0125] In a preferred embodiment of the present invention, the amplification primers are a composition of any multiple groups among nine groups of primers; in the 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):

[0126] (25.9 - 26.1):(3.9 - 4.1):(11.9 - 12.1).

[0127] Performing multiplex PCR amplification with the molar ratio of the above nine groups of primer sets 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 thus reducing the sequencing cost.

[0128] In a more preferred embodiment of the present invention, the molar ratio between the working concentrations of each primer set 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 = 3:2:2:9:17:11:26:4:12. Using multiple groups of amplification primers with the above molar ratio for multiplex PCR helps to improve the uniformity of amplification.

[0129] 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 - 10 base sequence. In a further preferred embodiment of the present invention, the tag sequence is a 6 - 8 base sequence.

[0130] 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 groups. 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 the typing results.

[0131] For simultaneous sequencing of multiple samples on a machine, amplification products with different tag sequences can be directly mixed and library constructed during 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. Amplification using amplification primers with tag sequences can improve the sequencing throughput of mixed library construction, and has the beneficial effects of being convenient, efficient and cost-reducing. The tag sequence can be any sequence combination that does not affect amplification and / or library construction operations.

[0132] In a specific 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.

[0133] 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.

[0134] In the second 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 an amplification product obtained by using the above-mentioned PCR amplification method for the HLA gene. Using the above-mentioned 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 genotyping.

[0135] In a preferred embodiment of the present invention, before ligating the above-mentioned amplification product with the above-mentioned sequencing adapter, the construction method further includes: purifying the above-mentioned 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-mentioned purification method is selected from any one or more of the following: magnetic bead method or silica gel column method.

[0136] In the third 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, improve the detection efficiency, and reduce the detection cost. The third-generation sequencing technology, with its characteristics of long read length and high throughput, can provide more comprehensive HLA gene information, thereby helping to improve the accuracy of HLA typing.

[0137] In the fourth 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.

[0138] In the fifth typical embodiment of the present invention, there is provided an application of the above-mentioned PCR amplification method of HLA gene, 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 detection.

[0139] 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.

[0140] The present invention will be further described in detail below with reference to specific embodiments, and these embodiments should not be construed as limiting the scope of the present invention claimed.

[0141] Example 1: Specificity of single primer and amplification system

[0142] I. PCR amplification

[0143] For different HLA gene targets, the designed primer sequences are shown in Table 1 below:

[0144] Table 1

[0145]

[0146]

[0147] 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 to 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 high polymorphism or complex gene structure, 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.

[0148] The PCR amplification system is shown in Table 2 below:

[0149] Table 2

[0150] Reagent Dosage / μL KOD FX Neo Buffer 12.5 dNTPs 5 KOD FX Neo 0.5 HLA-Primer 10μM 1 <![CDATA[H2O]]> 5 NA12878 gDNA 1

[0151] 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 the two downstream primers for HLA-DRB-E2 to 6 is F:R1:R2 = 2:1:1.

[0152] 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.

[0153] II. Purification

[0154] 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;

[0155] 2. Place it on the magnetic stand and let stand for 5 minutes until the magnetic beads are completely adsorbed, and remove the supernatant;

[0156] 3. Add 200 μL of freshly prepared 80% ethanol, let stand for 30 seconds, remove the supernatant, and repeat the washing once;

[0157] 4. Centrifuge briefly on a hand-held centrifuge to remove the residual alcohol;

[0158] 5. Resuspend with 20 μL of EB buffer and let stand at room temperature for 5 minutes;

[0159] 6. Place it on the magnetic stand and let stand for 3 minutes, then aspirate the supernatant to obtain the amplification product.

[0160] III. Detection

[0161] The amplified products were inspected for fragment distribution using an Agilent 2100 bioanalyzer.

[0162] Experimental results:

[0163] Figure 2 They were respectively the peak maps of the amplified 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).

[0164] Comparative Example 1

[0165] In this comparative example, the gene amplification primers in Patent Application CN 113817725 A were used for the experiment.

[0166] I. Amplification

[0167] The primer sequences are shown in Table 3 below:

[0168] Table 3

[0169]

[0170]

[0171] The PCR reaction system was the same as Table 2 in Example 1;

[0172] 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, increasing by 30 s for each cycle>) × 30 cycles, 68°C for 10 min, and stored at 4°C.

[0173] II. Purification and Detection

[0174] The purification steps and the method for detecting the fragment distribution of the amplified products were the same as those in Example 1.

[0175] Experimental results:

[0176] Figure 3Primers in the comparative example (SEQ ID NOs: 20 - 37): Amplification product peak diagrams 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).

[0177] Analysis of experimental results:

[0178] The experimental results showed that, compared with the amplification product peak diagrams of the corresponding target primers in Comparative Example 1 ( Figure 3 ), the non-specific amplification product peak signals in the amplification product peak diagrams of the primers designed for each target in Example 1 of the present invention ( Figure 2 ) were lower except for the main peaks, indicating that the specificity of the amplification of the primers designed for each target in Example 1 of the present invention was significantly improved.

[0179] Example 2: Multiplex primer amplification system and PCR procedure

[0180] I. Amplification

[0181] Using the primer sequences designed for different targets in Table 1, dilute with low TE buffer, and then mix according to the ratios 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.

[0182] Table 4

[0183]

[0184]

[0185] Configure the PCR amplification system as follows in Table 5:

[0186] Table 5

[0187] Reagent Dosage / μL KOD FX Neo Buffer 12.5 2mM dNTPs 5 KOD FX Neo 0.5 Primer Mix(10μM) 3 H2O 3 gDNA 1

[0188] The PCR reaction procedure is as follows:

[0189] PCR Program 1 (same as Example 1): 94°C × 2 min, (98°C × 10 s, 74°C × 12 min) × 4 cycles, (98°C × 10 s, 72°C × 12 min) × 4 cycles, (98°C × 10 s, 70°C × 12 min) × 4 cycles, (98°C × 10 s, 68°C × 12 min) × 18 cycles, 68°C × 7 min, hold at 4°C.

[0190] PCR Program 2: 94°C × 2 min, (98°C × 10 s, 72°C × 12 min) × 2 cycles, (98°C × 10 s, 70°C × 12 min) × 2 cycles, (98°C × 10 s, 68°C × 12 min) × 21 cycles, 68°C × 7 min, hold at 4°C.

[0191] PCR Program 3: 94°C × 2 min, (98°C × 10 s, 74°C 12 min) × 1 cycle, (98°C × 10 s, 73°C × 12 min) × 1 cycle, (98°C × 10 s, 72°C × 12 min) × 1 cycle, (98°C × 10 s, 71°C × 12 min) × 1 cycle, (98°C × 10 s, 70°C × 12 min) × 1 cycle, (98°C × 10 s, 69°C × 12 min) × 1 cycle, (98°C × 10 s, 68°C × 12 min) × 21 cycles, 68°C × 7 min, hold at 4°C.

[0192] PCR Program 4 (same as Comparative Example 1): 94°C × 2 min, (98°C × 10 s, 68°C × 12 min <starting from the 11th cycle, each cycle increases by 30 s>) × 30 cycles, 68°C × 10 min, hold at 4°C.

[0193] II. Purification

[0194] The purification steps are the same as those in Example 1.

[0195] III. Library Construction and Sequencing

[0196] The library was constructed according to the library construction process of the SMRTbell prep kit 3.0 from PacBio. The fragment distribution of the library was quality inspected using the Agilent Femto PμLse system. The on-machine sequencing was performed according to the on-machine process and reagents recommended by PacBio Revio.

[0197] Comparative Example 2: Multiple Primer Amplification

[0198] In this comparative example, the gene amplification primers in Patent Application CN 113817725 A were used for the experiment. The designed primer sequences are the same as those in Table 3 of Comparative Example 1.

[0199] Mix the primers in Table 3 according to the scheme in Table 6 below (named total primer). Among them, the molar ratio of the upstream and downstream primers targeting the same gene target is 1:1. The molar ratio of the upstream primer and the two downstream primers targeting HLA-DRB-E2 to 6 is F:R1:R2 = 2:1:1.

[0200] Table 6

[0201] Primer Name Added Ratio (Volume Ratio) HLA-DQA1-F&R&R(20μM) 1.8 HLA-DQB1-F&R(5μM) 1.1 HLA-DPA1-F&R(5μM) 0.3 HLA-DPB1-F&R(5μM) 1.1 HLA-A-F&R(5μM) 0.38 HLA-B-F&R(5μM) 0.2 HLA-C-F&R(5μM) 0.42 HLA-DRB-E1-F&R(5μM) 0.2 HLA-DRB-E2E6-F&R1&R2(5μM) 0.5

[0202] The PCR reaction system is shown in Table 7 below:

[0203] Table 7

[0204] Reagent Dosage / μL KOD FX Neo Buffer 12.5 dNTPs 5 KOD FX Neo 0.5 total primer 6 NA12878 gDNA 1

[0205] The PCR reaction program is the same as that in Comparative Example 1.

[0206] II. Purification

[0207] The purification steps are the same as those in Example 1.

[0208] III. Library construction and sequencing

[0209] The library construction and sequencing steps are the same as those in Example 2.

[0210] Experimental results: See Table 8, Table 9 and Figure 5 .

[0211] Table 8

[0212]

[0213] Table 9:

[0214] Example Capture Specificity (%) Example 2 - PCR Program 1 97％ Example 2 - PCR Program 2 97％ Example 2 - PCR Program 3 97％ Example 2 - PCR Program 4 93％ Comparative Example 2 89％

[0215] 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.

[0216] Analysis of experimental results:

[0217] 1) From the perspective of capture specificity

[0218] The results of Example 2 are shown in Table 8, Table 9 and Figure 5 As shown, in the same system, all 11 loci 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.

[0219] In addition, compared with the PCR programs without gradient annealing (Example 2 - PCR Program 4 and Comparative Example 2), the PCR program with a stepwise gradient annealing from high to low has higher capture specificity in amplification, indicating that gradient annealing significantly promotes amplification specificity. For example, Example 2 - PCR Program 1, Example 2 - PCR Program 2, and Example 2 - PCR Program 3.

[0220] Generally speaking, compared with Comparative Example 2, the target capture specificities of Example 2 - PCR Programs 1 to 4 designed in the present invention have all been improved, with the lowest specificity increase being 4 percentage points and the highest being 8 percentage points.

[0221] 2) From the perspective of the uniformity of sequencing depth

[0222] Compared with Comparative Example 2, the CV values (coefficient of variation) of the sequencing depths of each target in Example 2 - PCR Programs 1 - 4 of the present invention are lower, indicating better output uniformity of the present invention. Among them, the CV value of Example 2 - PCR Program 1 is the lowest and the uniformity is the best.

[0223] 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: at the 5.1% ratio of the lowest average depth of HLA - DRB - E2 - E6 in Example 2 - PCR Program 1, a minimum sequencing depth of 100× is required, and the total data volume depth to be measured is 1960× (the calculation formula is: 100×

[0224] / 5.1% = 1960×),

[0225] Similarly, when calculating at the 0.2% ratio 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

[0226] ×). It can be seen that the improved scheme of Example 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.

[0227] Example 3: Multiplex amplification system with barcode primers and genotyping detection

[0228] For different targets, the designed primer sequences are shown in Table 10 below (where the underlined NNNNNNNN represents the barcode sequence, and the rest are primer sequences):

[0229] Table 10

[0230]

[0231]

[0232] The barcode sequences used in this experiment are as follows:

[0233] Table 11

[0234] Sample Forward and Reverse barcode sequences NA17052 CGGTCATA NA17078 AGTCGCTT NA17130 ACAGCAAC NA17248 CCTCAGTT NA17288 ATTCGAGG NA17295 AACTGGTG

[0235] The primers were diluted with low TE buffer and then mixed according to the ratio in Example 2, named primermix;

[0236] Configure the PCR amplification system according to Example 2; the PCR reaction procedure is the same as that in Example 1.

[0237] II. Purification

[0238] The purification steps are the same as those in Example 1.

[0239] III. Library construction and sequencing

[0240] The above 6 groups of PCR products were mixed in equal mass, and the library was constructed according to the library construction process of the PacBio SMRTbell prep kit 3.0. The quality inspection of the library for fragment distribution was performed using the Agilent Femto Pulse system. The on-machine sequencing was operated according to the on-machine process recommended by PacBio Revio.

[0241] IV. Compare the sequencing data with the data recorded in the IMGT HLA database to analyze and determine the specific typing results.

[0242] Experimental results:

[0243] Six samples were amplified and mixed for library construction using six 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 of the mixed library construction after amplification with barcode primers is normal.

[0244] Table 12

[0245] Sample Output Data Volume (bp) Proportion of Output Data NA17052 391708562 18％ NA17078 350156943 16％ NA17130 356546874 17％ NA17248 363522155 17％ NA17288 351583836 16％ NA17295 336733018 16％

[0246] As can be seen from Table 12, regarding the results of the mixed library construction and sequencing data splitting of the six samples obtained by using the scheme designed in the present invention, the present invention has good library construction sequencing data yield and uniformity.

[0247] Table 13

[0248]

[0249] (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.)

[0250] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0251] The present invention designs primers for 11 targets of HLA to achieve near-full-length amplification of HLA genes. The amplification products are sequenced using a third-generation sequencing platform to obtain the full-length sequences of HLA genes, improving the resolution of HLA typing. It can achieve 6-digit typing with 100% accuracy, and the 11-target amplification primers are in the same reaction system to achieve one-tube detection.

[0252] In terms of primer design, the present invention adjusts the sequence length of the amplification primers to make the annealing temperatures of the amplification primers uniform, 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.

[0253] The present invention can improve the amplification uniformity of primers and thus improve the data utilization rate by adjusting the usage ratio between primers.

[0254] Barcodes are added to the primer ends, and different barcodes are used for amplification of different samples. 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.

[0255] 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 modification, equivalent replacement, improvement, 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 PCR amplification method for HLA genes, characterized in that, The method includes amplifying the HLA gene using a PCR amplification program as shown in the following formula; the formula is: Among them, in the annealing cycle, the temperature T of annealing extension n is from T start to T end with a gradient decrease, and the decreased temperature gradient is ΔT. N stage cycles are carried out at each annealing temperature; in the end preservation, it is continuously kept at a constant temperature at the temperature T 恒温 . The said T n = T start - n·ΔT (n = 0, 1, 2, …), where T start ≥ T n ≥ T end ; The T start is 72°C to 74°C; the T end is 66°C to 70°C; the ΔT is 0.5°C to 2°C; where H is 10 min to 18 min; The said where N 总 is 25 to 35 cycles; The said N stage is from 1 to 6 cycles; T0 is 94°C to 98°C; H0 is 30 s to 5 min; T1 is 94°C to 98°C; H1 is 10 s to 1 min; H2 is 5 min to 10 min.

2. The amplification method according to claim 1, wherein The said T end is 68 °C; Optionally, H is 10 min to 15 min; Optionally, the N 总 is 25 to 30 cycles; Optionally, said N stage is 1 to 4 cycles; Optionally, ΔT is 1°C to 2°C; Optionally, H is 12 min; Optionally, T0 is 94°C; Optionally, H0 is 2 min to 5 min; Optionally, H0 is 2 min; Optionally, T1 is 98°C; Optionally, H1 is 10 s; Optionally, H2 is 7 min; Optionally, the T 恒温 is 4°C to 25°C Optionally, the T 恒温 is 4 °C.

3. The amplification method according to claim 2, wherein The method includes: The reaction program of the PCR amplification is selected from any one of the following: 1) 94°C × 2 min, (98°C × 10 s, 74°C × 12 min) × 4 cycles, (98°C × 10 s, 72°C × 12 min) × 4 cycles, (98°C × 10 s, 70°C × 12 min) × 4 cycles, (98°C × 10 s, 68°C × 12 min) × 18 cycles, 68°C × 7 min, 4°C × constant temperature preservation; 2) 94°C × 2 min, (98°C × 10 s, 72°C × 12 min) × 2 cycles, (98°C × 10 s, 70°C × 12 min) × 2 cycles, (98°C × 10 s, 68°C × 12 min) × 21 cycles, 68°C × 7 min, 4°C × constant temperature preservation; 3) 94°C × 2 min, (98°C × 10 s, 74°C 12 min) × 1 cycle, (98°C × 10 s, 73°C × 12 min) × 1 cycle, (98°C × 10 s, 72°C × 12 min) × 1 cycle, (98°C × 10 s, 71°C × 12 min) × 1 cycle, (98°C × 10 s, 70°C × 12 min) × 1 cycle, (98°C × 10 s, 69°C × 12 min) × 1 cycle, (98°C × 10 s, 68°C × 12 min) × 21 cycles, 68°C × 7 min, 4°C × constant temperature preservation.

4. The amplification method according to claim 1, wherein The method uses a composition containing any one or any combination of the following nine sets of primers: Among them, The first set of primers includes 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; The second set of primers includes 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; The third set of primers includes 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; The fourth set of primers includes 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; The fifth set 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; The sixth set 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; The seventh set 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; The eighth set 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; 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.

5. The amplification method according to claim 4, wherein Among the nine sets of primers, the molar ratio between the upstream primer and the downstream primer within each set of primers is: (0.9 - 1.1):(0.9 - 1.1); The amplification primers are a composition of any multiple sets among the 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).

6. The amplification method according to claim 4, wherein The 5'-end of the amplification primers contains a tag sequence; the tag sequence is a 6 - 10 bp oligonucleotide sequence.

7. A method for constructing an HLA gene sequencing library, characterized in that, The construction method comprises: connecting the amplification product of the HLA gene with a sequencing adapter to obtain the HLA gene sequencing library; The amplification product is an amplification product amplified by the amplification method described in any one of claims 1 - 6.

8. A method for HLA gene sequencing, characterized in that, The HLA gene sequencing method comprises: sequencing the sequencing library; the sequencing library is a sequencing library constructed by the construction method of the HLA gene sequencing library described in claim 7; Optionally, the sequencing uses third-generation sequencing technology.

9. A method for HLA genotyping detection, characterized in that, The HLA gene typing detection method comprises: detecting and typing the HLA genotype by using the HLA gene sequencing method described in claim 8.

10. Use of the PCR amplification method of the HLA gene described in any one of claims 1 - 6, or the construction method of the HLA gene sequencing library described in claim 7, or the HLA gene sequencing method described in claim 8, or the HLA gene typing detection method described in claim 9 in HLA gene typing detection; Optionally, the HLA gene typing detection sequentially comprises the following steps: HLA gene amplification, HLA gene sequencing library construction, HLA gene sequencing, and HLA gene typing determination.

Images