HLA-DRB1 gene high-resolution typing method based on group specific primers

Abstract

The invention discloses an HLA-DRB1 gene high-resolution typing method based on group specific primers, and belongs to the technical field of gene detection. The group of specific primers are respectively designed according to exons 1-4 of the HLA-DRB1 gene, sequences of the specific primers are shown as SEQ ID NO.1-SEQ ID NO.20, the specific primers have good conservative property on allele sequences of the HLA-DRB1 gene, compared with gene sequences of HLA-DRB3, HLA-DRB4 and HLA-DRB5, the specific primers have good specificity, 1, 2, 3 and 4 exons of the HLA-DRB1 site can be effectively amplified, the corresponding exons are sequenced, and the specific primers can be used for detecting the HLA-DRB1 gene. The polymorphic site of the HLA-DRB1 gene is fully covered. Therefore, when further typing is carried out, the resolution and accuracy of HLA-DRB1 genetic typing are effectively improved, and the ambiguous condition of HLA-DRB1 genetic typing is improved.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and in particular relates to a high-resolution HLA-DRB1 gene typing method based on group-specific primers. Background Technology

[0002] The human leukocyte antigen (HLA) complex is located on the short arm of human chromosome 6 (6p21.3). Composed of a group of closely linked genes, it is the most complex and polymorphic genetic system known to humankind. Its expression products are important antigens contributing to transplant rejection and are closely related to human immune function. HLA gene detection methods include serological typing, cytological typing, and DNA typing. With the rapid development of molecular genetics and gene manipulation techniques, DNA typing has become the primary detection method.

[0003] Polymerase chain reaction-direct sequencing (PCR-SBT) is an HLA typing method based on first-generation sequencing. It is also the WHO-recommended high-resolution HLA genotyping method and is currently the "gold standard" for high-resolution HLA typing. It has high sensitivity, can directly obtain DNA sequences, detect new alleles, and identify all existing polymorphisms, making it the most direct and intuitive method.

[0004] The HLA-DRB1 gene is a gene within the HLA-DR locus, consisting of six exons and approximately 13.4 kb in length. Its polymorphism is primarily concentrated in exons 2 and 3. The HLA-DRB1 gene exhibits high polymorphism; currently, the IPD-IMGT / HLA database lists nearly 4000 alleles for HLA-DRB1, making it the gene with the most alleles among HLA class II genes. The current standard PCR-SBT method for HLA-DRB1 gene typing involves amplifying and resequencing exons 2 and 3, or amplifying and resequencing only exon 2. However, due to the extremely high polymorphism of the HLA-DRB1 gene and the difficulty in finding suitable conserved regions, this method significantly increases the complexity of primer design and amplification. Meanwhile, although the polymorphic sites of the HLA-DRB1 gene are mainly located in exons 2 and 3, there are still many allele differences located outside exons 2 and 3, which increases the possibility of ambiguity in HLA-DRB1 gene typing.

[0005] Next-generation sequencing (NGS) and third-generation sequencing (TGS) are two other HLA genotyping techniques. Compared to PCR-SBT, NGS can effectively reduce the occurrence of ambiguous results, but a small number still occur; while third-generation sequencing is very expensive, limiting its application. Therefore, there is an urgent need to develop new technologies that can effectively improve the resolution and accuracy of HLA-DRB1 genotyping. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, this invention provides a high-resolution HLA-DRB1 gene genotyping method based on group-specific primers. The method detects exons 1 to 4 of the HLA-DRB1 gene. The primers exhibit good conservation across all allele sequences of the HLA-DRB1 gene and demonstrate good specificity compared to the HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes. This effectively amplifies exons 1, 2, 3, and 4 of the HLA-DRB1 locus, and the corresponding exons are sequenced, fully covering the polymorphic sites of the HLA-DRB1 gene. Therefore, in further genotyping, this method effectively improves the resolution and accuracy of HLA-DRB1 gene genotyping, mitigating ambiguity in HLA-DRB1 gene genotyping.

[0007] The basic principle of this invention is that the HLA-DRB1 gene is the gene with the most alleles among HLA class II genes, with nearly 4,000 alleles in the IPD-IMGT / HLA database. It has high polymorphism. Therefore, it is necessary to design group-specific primers that can amplify and re-sequencing HLA-DRB1 exons 1, 2, 3, and 4 in a limited conserved region for genotyping.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a primer set for high-resolution HLA-DRB1 gene genotyping. The primer set includes group-specific primers designed for exons 1-4 of the HLA-DRB1 gene, and the HLA-DRB1 gene is amplified by PCR using these group-specific primers. The sequence of the group-specific primer designed for exon 1 of the HLA-DRB1 gene is shown in SEQ ID NO. 1-SEQ ID NO. 4; the sequence of the group-specific primer designed for exon 2 of the HLA-DRB1 gene is shown in SEQ ID NO. 5-SEQ ID NO. 10; the sequence of the group-specific primer designed for exon 3 of the HLA-DRB1 gene is shown in SEQ ID NO. 11-SEQ ID NO. 15; and the sequence of the group-specific primer designed for exon 4 of the HLA-DRB1 gene is shown in SEQ ID NO. 16-SEQ ID NO. 20.

[0009] Furthermore, The group-specific primers designed based on HLA-DRB1 gene exon 1 include: DRB1 The amplification primer sequences for the 01 / 15 / 16 alleles are shown in SEQ ID NO.1 and SEQ ID NO.2; DRB1 The amplification primer sequences for the 03 / 08 / 10 / 11 / 12 / 13 / 14 alleles are shown in SEQ ID NO.1 and SEQ ID NO.3: DRB1 The primer sets for amplification of the 04 / 07 / 09 alleles are shown in SEQ ID NO.1 and SEQ ID NO.4; The group-specific primers designed based on HLA-DRB1 gene exon 2 include: DRB1 The amplification primer sequences for alleles 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.5 and SEQ ID NO.6; DRB1 The amplification primer sequences for the 07 / 09 alleles are shown in SEQ ID NO.7 and SEQ ID NO.8; DRB1 The amplification primer sequences for the 10 alleles are shown in SEQ ID NO.9 and SEQ ID NO.10; The group-specific primers designed based on HLA-DRB1 gene exon 3 include: DRB1 The amplification primer sequences for alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.11 and SEQ ID NO.12; DRB1 The amplification primer sequences for the 04 / 07 / 09 allele are shown in SEQ ID NO.13 and SEQ ID NO.14; DRB1 The amplification primer sequences for the 10 alleles are shown in SEQ ID NO.11 and SEQ ID NO.15; The group-specific primers designed based on exon 4 of the HLA-DRB1 gene include: DRB1 The amplification primer sequences for alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.16 and SEQ ID NO.17; DRB1 The amplification primer sequences for the 04 / 07 / 09 alleles are shown in SEQ ID NO.18 and SEQ ID NO.19; DRB1 The primer sequences for amplification of the 10 alleles are shown in SEQ ID NO.20 and SEQ ID NO.19.

[0010] A second objective of this invention is to provide the application of any of the primer sets described above in the preparation of high-resolution HLA-DRB1 gene typing products, wherein the method for high-resolution HLA-DRB1 gene typing includes the following steps: (1) Extract genomic DNA from the sample to be tested and dilute it to 10 ng / μl according to the DNA quantification results; (2) The DNA of the test sample was amplified by PCR using the group-specific amplification primers to obtain amplification products containing exons 1, 2, 3 and 4 of the HLA-DRB1 gene, respectively. (3) Perform agarose gel electrophoresis on the amplification products; (4) Sequencing the PCR products using sequencing primers; (5) The sequencing results were compared with the standard exon sequences of the HLA-DRB1 gene in the IMGT / HLA database to finally determine the type of the HLA-DRB1 gene.

[0011] Furthermore, the amplification system in step (2) is 10 μl, containing 5 μl of 2×Taq Master mix or 2×HotStart Taq Master mix, 0.4 μl of F primer, 0.4 μl of R primer, 0.5 μl of template DNA, and ddH2O added to 10 μl.

[0012] Furthermore, The 2×Taq Master mix is ​​used to amplify DRB1 in exon 1. 01 / 15 / 16 alleles, DRB1 Alleles 03 / 08 / 10 / 11 / 12 / 13 / 14; DRB1 in exon 2 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 Alleles, DRB1 07 / 09 alleles, DRB1 10 alleles; DRB1 in exon 3 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles; and DRB1 in exon 4 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles; The 2×HotStart Taq Master mix is ​​used to amplify DRB1 in exon 1. 04 / 07 / 09 alleles.

[0013] Furthermore, in step (2): HLA-DRB1 gene exon 1 DRB1 01 / 15 / 16 alleles and DRB1 The reaction conditions for allele PCR amplification on March 8th, 10th, 11th, 12th, 13th, and 14th were: 94℃, 3 min; 94℃, 20 s → 61℃, 20 s → 72℃, 40 s (35 cycles); 72℃, 5 min; for HLA-DRB1 gene exon 1 DRB1. The reaction conditions for allele PCR amplification on April 7th and September 2009 were as follows: 94℃, 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 63℃, 20 s → 72℃, 40 s (29 cycles); 72℃, 5 min. HLA-DRB1 gene exon 2 DRB1 The reaction conditions for allele PCR amplification on January 1st, 3rd, 4th, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 72℃ for 20 seconds → 72℃ for 40 seconds (10 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 seconds → 67℃ for 20 seconds → 72℃ for 40 seconds (25 cycles); 72℃ for 5 minutes; for exon 2 DRB1 of the HLA-DRB1 gene. The reaction conditions for PCR amplification of the 07 / 09 alleles were: 94℃ for 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 65℃, 20 s → 72℃, 40 s (31 cycles); 72℃ for 5 min; targeting exon 2 DRB1 of the HLA-DRB1 gene. The reaction conditions for PCR amplification of 10 alleles were 94℃ for 3 min; 94℃ for 20 s → 70℃ for 20 s → 72℃ for 30 s (35 cycles); 72℃ for 5 min. HLA-DRB1 gene exon 3 DRB1 The reaction conditions for allele PCR amplification on January 1st, 3rd, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 63℃ for 20 seconds → 72℃ for 40 seconds (35 cycles); 72℃ for 5 minutes; for exon 3 of the HLA-DRB1 gene, DRB1. The reaction conditions for allele PCR amplification on April 7th, 2009 were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (35 cycles); 72℃ for 5 min; targeting exon 3 of the HLA-DRB1 gene, DRB1. The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (33 cycles); 72℃ for 5 min. HLA-DRB1 gene exon 4 DRB1 The reaction conditions for allele PCR amplification on January 1, 3, 8, 11, 12, 13, 14, 15, and 16 were: 94℃ for 3 min; 94℃ for 20 s → 63℃ for 20 s → 72℃ for 40 s (35 cycles); 72℃ for 5 min; for exon 4 DRB1 of the HLA-DRB1 gene. The reaction conditions for allele PCR amplification on April 7th, 2009 were: 94℃ for 3 min; 94℃, 20 s → 64℃, 20 s → 72℃, 60 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 60℃, 20 s → 72℃, 60 s (27 cycles); 72℃ for 5 min; targeting exon 4 of the HLA-DRB1 gene, DRB1. The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (31 cycles); 72℃ for 5 min.

[0014] Furthermore, in step (3), 2% agarose gel is used, the voltage is 120V, and the time is 30min.

[0015] Furthermore, in step (4): The sequencing primer sequences for all alleles of exon 1 of the HLA-DRB1 gene are shown in SEQ ID NO.1; HLA-DRB1 gene exon 2 DRB1 The sequencing primer sequences for alleles 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO. 5, DRB1. The sequencing primer sequences for the 07 / 09 alleles are shown in SEQ ID NO.7, DRB1 The sequencing primer sequences for the 10 alleles are shown in SEQ ID NO. 9; HLA-DRB1 gene exon 3 DRB1 The sequencing primer sequences for the alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.11 and SEQ ID NO.12, DRB1 The sequencing primer sequences for the 04 / 07 / 09 alleles are shown in SEQ ID NO.13, DRB1. The sequencing primer sequences for the 10 alleles are shown in SEQ ID NO.15; The sequencing primer sequences for all alleles of exon 4 of the HLA-DRB1 gene are shown in SEQ ID NO.19.

[0016] A third objective of this invention is to provide a kit for high-resolution HLA-DRB1 gene typing, wherein the kit contains any of the primer sets described above.

[0017] Furthermore, the kit also contains sequencing primers used in any of the above-described applications.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a high-resolution HLA-DRB1 gene genotyping method based on group-specific primers. This method detects exons 1 to 4 of the HLA-DRB1 gene. The primers exhibit good conservation across all allele sequences of the HLA-DRB1 gene and show good specificity compared to the HLA-DRB3, HLA-DRB4, and HLA-DRB5 gene sequences. This effectively amplifies exons 1, 2, 3, and 4 of the HLA-DRB1 locus, and the corresponding exons are sequenced, fully covering the polymorphic sites of the HLA-DRB1 gene. Therefore, in further genotyping, this method effectively improves the resolution and accuracy of HLA-DRB1 gene genotyping, addressing ambiguities in HLA-DRB1 gene genotyping. Attached Figure Description

[0019] Figure 1 This is an electrophoresis image of the group-specific primer amplification products of HLA-DRB1 gene exon 1 in Example 1 (6 samples). Figure 1 A: Amplify DRB1 Agarose gel electrophoresis images of PCR products of alleles from January 15th to 16th; Figure 1 B: Amplification of DRB1 Agarose gel electrophoresis images of allele PCR products from 03 / 08 / 10 / 11 / 12 / 13 / 14; Figure 1 C: Amplification of DRB1 Agarose gel electrophoresis image of allele PCR products from 04 / 07 / 09; Figure 2 This is an electrophoresis image of the group-specific primer amplification products of HLA-DRB1 gene exon 2 in Example 1 (6 samples). Figure 2 A: Amplify DRB1 Agarose gel electrophoresis images of allele PCR products from 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16. Figure 2 B: Amplification of DRB1 Agarose gel electrophoresis image of PCR products of the 07 / 09 alleles; Figure 2 C: Amplification of DRB1 Agarose gel electrophoresis images of PCR products from 10 alleles; Figure 3 This is an electrophoresis image of the group-specific primer amplification products of HLA-DRB1 gene exon 3 in Example 1 (6 samples). Figure 3 A: Amplify DRB1 Agarose gel electrophoresis images of allele PCR products from 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16; Figure 3 B: Amplification of DRB1 Agarose gel electrophoresis image of allele PCR products from 04 / 07 / 09; Figure 3 C: Amplification of DRB1 Agarose gel electrophoresis images of PCR products from 10 alleles; Figure 4 This is an electrophoresis image of the group-specific primer amplification products of HLA-DRB1 gene exon 4 in Example 1 (6 samples). Figure 4 A: Amplify DRB1 Agarose gel electrophoresis images of allele PCR products from 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16; Figure 4 B: Amplification of DRB1 Agarose gel electrophoresis image of allele PCR products from 04 / 07 / 09; Figure 4 C: Amplification of DRB1 Agarose gel electrophoresis images of PCR products from 10 alleles; Figure 5 This is the sequencing diagram of exon 1 of the HLA-DRB1 gene in sample 1 of Example 1; Figure 6 This is the sequencing diagram of exon 2 of the HLA-DRB1 gene in sample 1 of Example 1; Figure 7 This is the sequencing diagram of exon 3 of the HLA-DRB1 gene in sample 1 of Example 1; Figure 8 This is the sequencing diagram of exon 4 of the HLA-DRB1 gene in sample 1 of Example 1. Detailed Implementation

[0020] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0021] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0022] Example 1 The purpose of this embodiment is to use group-specific primers to perform high-resolution genotyping of the HLA-DRB1 locus in 6 human blood samples.

[0023] In this embodiment, the primer orientations are all from 5' to 3', and the HLA-DRB1 gene template is DRB1. 03010101, all allele sequences are referenced from the IMGT / HLA professional website database: http: / / www.ebi.ac.uk / imgt / hla / index.html.

[0024] The specific steps of the method are as follows: (1) Extraction of genomic DNA Genomic DNA was extracted using the Axygen DNA Extraction Kit (Axygen Biotechnology (Hangzhou) Co., Ltd.).

[0025] (2) PCR amplification The genomic DNA extracted in step (1) was amplified using group-specific primers. Wherein: ①The exon 1 specific primers for the HLA-DRB1 gene include: DRB1 Amplification primers for 01 / 15 / 16: DRB1-F1-1 (SEQ ID NO.1): 5'-CATAGTTCTCCCTGAGTGAGAC-3'; DRB1-R1-1 (SEQ ID NO.2): 5'-AAGGTCCTGTGGGGAACCTA-3'; DRB1 Amplification primers for 03 / 08 / 10 / 11 / 12 / 13 / 14: DRB1-F1-1 (SEQ ID NO.1): 5'-CATAGTTCTCCCTGAGTGAGAC-3'; DRB1-R1-2 (SEQ ID NO.3): 5'-GGTGACATAACCCAGGATCGTTT-3'; DRB1 Amplification primers for 04 / 07 / 09: DRB1-F1-1 (SEQ ID NO.1): 5'-CATAGTTCTCCCTGAGTGAGAC-3'; DRB1-R1-3 (SEQ ID NO.4): 5'-TGTGGAGGACAACACAGGATCG-3'; ②The exon 2 specific primers for the HLA-DRB1 gene include: DRB1 Amplification primers for 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16: DRB1-F2-1 (SEQ ID NO.5): 5'-GGAGGTCTCCAGAACAGGCT-3'; DRB1-R2-1 (SEQ ID NO.6): 5'-CTGCCTCCCCTCCCACAACA-3'; DRB1 07 / 09 Amplification Primers: DRB1-F2-2 (SEQ ID NO.7): 5'-GATCCTCCTCCAGCTCCTGC-3'; DRB1-R2-2 (SEQ ID NO.8): 5'-CCAGGACAATCAAGGTCCAATCTG-3'; DRB1 10 Amplification Primers: DRB1-F2-3 (SEQ ID NO.9): 5'-GGGCGGGTTAAGGTTCCAGT-3'; DRB1-R2-3 (SEQ ID NO.10): 5'-CACACACACAGAGTCAGATTCCCAGC-3'; ③ The group-specific primers for exon 3 of the HLA-DRB1 gene include: DRB1 Amplification primers for 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16: DRB1-F3-1 (SEQ ID NO. 11): 5'-GGTTTGCAGAGAAGCAGACA-3'; DRB1-R3-1 (SEQ ID NO.12): 5'-AGACTGCTTCTCCAGGAGGTA-3'; DRB1 Amplification primers for 04 / 07 / 09: DRB1-F3-2 (SEQ ID NO. 13): 5'-AGCCTAGGTTTTACAGAGAAGCA-3'; DRB1-R3-2 (SEQ ID NO. 14): 5'-CAGGTGTTTCTAGAAAGTATTACAGGG-3'; DRB1 10 Amplification Primers: DRB1-F3-1 (SEQ ID NO. 11): 5'-GGTTTGCAGAGAAGCAGACA-3'; DRB1-R3-3 (SEQ ID NO.15): 5'-TTTCTAGAAACTATTACAGGACTATCCC-3'; ④ The group-specific primers for exon 4 of the HLA-DRB1 gene include: DRB1 Amplification primers for 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16: DRB1-F4-1 (SEQ ID NO.16): 5'-ACCTCCTGGAGAAGCAGTCT-3'; DRB1-R4-1 (SEQ ID NO. 17): 5'-TGTCTGTCACAGGATCCAGTGTAA-3'; DRB1 Amplification primers for 04 / 07 / 09: DRB1-F4-2 (SEQ ID NO.18): 5'-CCTGTAATACTTTCTAGAAACACCTGTAC-3'; DRB1-R4-2 (SEQ ID NO. 19): 5'-AGGCCACAAGCTATTATGCTTT-3'; DRB1 10 Amplification Primers: DRB1-F4-3 (SEQ ID NO.20): 5'-GGGGATAGTCCTGTAATAGTTTC-3'; DRB1-R4-2 (SEQ ID NO. 19): 5'-AGGCCACAAGCTATTATGCTTT-3'; The PCR reaction system consisted of 10 μl, and its composition is shown in Table 1 below: Table 1 PCR reaction system

[0026] The Mix (2×) is either 5 μl of 2×Taq Master mix or 2×HotStart Taq Master mix. The 2×Taq Master mix is ​​used to amplify DRB1 in exon 1. 01 / 15 / 16 alleles, DRB1 Alleles 03 / 08 / 10 / 11 / 12 / 13 / 14, DRB1 in exon 2 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 Alleles, DRB1 07 / 09 alleles, DRB1 10 alleles, DRB1 in exon 3 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles, DRB1 in exon 4 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles. 2×HotStart Taq Master mix for amplifying exon 1 DRB1. 04 / 07 / 09 alleles.

[0027] The PCR amplification conditions are as follows: ① DRB1 of exon 1 of the HLA-DRB1 gene 01 / 15 / 16 alleles and DRB1 The reaction conditions for allele PCR amplification on March / August / October / November / December / 13 / 14 were as follows: 94℃, 3 min; 94℃, 20 s → 61℃, 20 s → 72℃, 40 s (35 cycles); 72℃, 5 min. DRB1 in exon 1 The reaction conditions for PCR amplification of the 04 / 07 / 09 alleles were: 94℃, 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 63℃, 20 s → 72℃, 40 s (29 cycles); 72℃, 5 min.

[0028] ② DRB1 in exon 2 of the HLA-DRB1 gene The reaction conditions for allele PCR amplification on January 1st, 3rd, 4th, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds, then 72℃ for 20 seconds, then 72℃ for 40 seconds (10 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 seconds, then 67℃ for 20 seconds, then 72℃ for 40 seconds (25 cycles); 72℃ for 5 minutes. DRB1 in exon 2... The reaction conditions for PCR amplification of the 07 / 09 alleles were: 94℃ for 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 65℃, 20 s → 72℃, 40 s (31 cycles); 72℃ for 5 min. DRB1 in exon 2... The reaction conditions for PCR amplification of 10 alleles were 94℃ for 3 min; 94℃ for 20 s → 70℃ for 20 s → 72℃ for 30 s (35 cycles); 72℃ for 5 min.

[0029] ③ DRB1 in exon 3 of the HLA-DRB1 gene The reaction conditions for allele PCR amplification on January 1st, 3rd, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 63℃ for 20 seconds → 72℃ for 40 seconds (35 cycles); 72℃ for 5 minutes. DRB1 in exon 3... The PCR amplification conditions for the alleles on April 7th and 9th were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (35 cycles); 72℃ for 5 min. DRB1 in exon 3... The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (33 cycles); 72℃ for 5 min.

[0030] ④ DRB1 in exon 4 of the HLA-DRB1 gene The reaction conditions for allele PCR amplification on January 1st, 3rd, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 63℃ for 20 seconds → 72℃ for 40 seconds (35 cycles); 72℃ for 5 minutes. DRB1 in exon 4... The PCR amplification conditions for alleles on April 7th and 9th were: 94℃ for 3 min; 94℃, 20 s → 64℃, 20 s → 72℃, 60 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 60℃, 20 s → 72℃, 60 s (27 cycles); 72℃ for 5 min. DRB1 in exon 4... The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (31 cycles); 72℃ for 5 min.

[0031] (3) Agarose gel electrophoresis The amplified PCR products were subjected to electrophoresis on a 2% agarose gel to determine whether the target gene had been amplified. The voltage was 120V and the time was 30min.

[0032] Electrophoresis diagram of the amplified PCR products is shown below. Figure 1-4 As shown. Based on all electrophoresis results, it is indicated that the allele in sample 1 is DRB1. 04 and DRB1 07 / 09, Sample 2 allele is DRB1 03 / 08 / 11 / 12 / 13 / 14 and DRB1 04, Sample 3 allele is DRB1 03 / 08 / 11 / 12 / 13 / 14 and DRB1 03 / 08 / 11 / 12 / 13 / 14, Sample 4 allele is DRB1 01 / 15 / 16 and DRB1 On March 8th, 2011, 2012, 2013, and 2014, sample 5 had the DRB1 allele. 03 / 08 / 11 / 12 / 13 / 14 and DRB1 04, Sample 6 allele is DRB1 03 / 08 / 11 / 12 / 13 / 14 and DRB1 04.

[0033] (4) DNA sequencing The PCR products were sent to the company for sequencing. The sequencing peak diagram of exons 1-4 of sample 1 is shown below. Figure 5-8 As shown. The specific sequencing primers used are as follows: ①The HLA-DRB1 gene exon 1 sequencing primers include: All allele sequencing primers: DRB1-SF1 (SEQ ID NO.1): 5'-CATAGTTCTCCCTGAGTGAGAC-3'; ②The HLA-DRB1 gene exon 2 sequencing primers include: DRB1 Sequencing primers for 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16: DRB1-SF2-1 (SEQ ID NO.5): 5'-GGAGGTCTCCAGAACAGGCT-3'; DRB1 07 / 09 Sequencing primers: DRB1-SF2-2 (SEQ ID NO.7): 5'-GATCCTCCTCCAGCTCCTGC-3'; DRB1 10 sequencing primers: DRB1-SF2-3 (SEQ ID NO.9): 5'-GGGCGGGTTAAGGTTCCAGT-3'; ③The HLA-DRB1 gene exon 3 sequencing primers include: DRB1 Sequencing primers for 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16: DRB1-SF3-1 (SEQ ID NO.11): 5'-GGTTTGCAGAGAAGCAGACA-3'; DRB1-SR3-1 (SEQ ID NO.12): 5'-AGACTGCTTCTCCAGGAGGTA-3'; DRB1 Sequencing primers for 04 / 07 / 09: DRB1-SF3-2 (SEQ ID NO.13): 5'-AGCCTAGGTTTTACAGAGAAGCA-3'; DRB1 10 sequencing primers: DRB1-SR3-2 (SEQ ID NO.15): 5'-TTTCTAGAAACTATTACAGGACTATCCC-3'; ④ The HLA-DRB1 gene exon 4 sequencing primers include: All allele sequencing primers: DRB1-SR4 (SEQ ID NO. 19): 5'-AGGCCACAAGCTATTATGCTTT-3'; (5) Result judgment The sequencing results of 6 samples were evaluated using the method of this invention, and compared with the sequencing results evaluated based only on exon 2 and the sequencing results evaluated based only on exon 2 and exon 3. The results are shown in Table 2.

[0034] Table 2 Details of result judgment for different methods

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] The above results demonstrate that the high-resolution HLA-DRB1 gene genotyping method based on group-specific primers of this invention detects exons 1 to 4 of the HLA-DRB1 gene. The primers exhibit good conservation across all allele sequences of the HLA-DRB1 gene and show good specificity compared to the HLA-DRB3, HLA-DRB4, and HLA-DRB5 gene sequences. This method effectively amplifies exons 1, 2, 3, and 4 of the HLA-DRB1 locus, and the corresponding exons are sequenced, fully covering the polymorphic sites of the HLA-DRB1 gene. Therefore, in further genotyping, it avoids the problem of ineffective genotyping when certain allele nucleotides are located outside the amplified region, effectively improving the resolution and accuracy of HLA-DRB1 gene genotyping and resolving ambiguities in HLA-DRB1 gene genotyping.

[0044] While some existing patents disclose HLA genotyping products or methods, some only detect exon 2, while others only detect exon 2 and exon 3. Under current conditions, the products in these patents cannot yield accurate results. Some patents use third-generation sequencing to analyze HLA-DRB1, which includes all exons of the HLA-DRB1 gene, but this involves numerous steps and is costly.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer set for high-resolution HLA-DRB1 gene typing, characterized in that, The method includes group-specific primers designed based on exons 1-4 of the HLA-DRB1 gene, and uses these group-specific primers to perform PCR amplification of the HLA-DRB1 gene. The sequences of the group-specific primers designed based on exon 1 of the HLA-DRB1 gene are shown in SEQ ID NO.1-SEQ ID NO.4; the sequences of the group-specific primers designed based on exon 2 of the HLA-DRB1 gene are shown in SEQ ID NO.5-SEQ ID NO.10; the sequences of the group-specific primers designed based on exon 3 of the HLA-DRB1 gene are shown in SEQ ID NO.11-SEQ ID NO.15; and the sequences of the group-specific primers designed based on exon 4 of the HLA-DRB1 gene are shown in SEQ ID NO.16-SEQ ID NO.

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2. The primer set according to claim 1, characterized in that, The group-specific primers designed based on HLA-DRB1 gene exon 1 include: DRB1 The amplification primer sequences for the 01 / 15 / 16 alleles are shown in SEQ ID NO.1 and SEQ ID NO.2; DRB1 The amplification primer sequences for the 03 / 08 / 10 / 11 / 12 / 13 / 14 alleles are shown in SEQ ID NO.1 and SEQ ID NO.3: DRB1 The primer sets for amplification of the 04 / 07 / 09 alleles are shown in SEQ ID NO.1 and SEQ ID NO.4; The group-specific primers designed based on HLA-DRB1 gene exon 2 include: DRB1 The amplification primer sequences for alleles 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.5 and SEQ ID NO.6; DRB1 The amplification primer sequences for the 07 / 09 alleles are shown in SEQ ID NO.7 and SEQ ID NO.8; DRB1 The amplification primer sequences for the 10 alleles are shown in SEQ ID NO.9 and SEQ ID NO.10; The group-specific primers designed based on HLA-DRB1 gene exon 3 include: DRB1 The amplification primer sequences for alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.11 and SEQ ID NO.12; DRB1 The amplification primer sequences for the 04 / 07 / 09 allele are shown in SEQ ID NO.13 and SEQ ID NO.14; DRB1 The amplification primer sequences for the 10 alleles are shown in SEQ ID NO.11 and SEQ ID NO.15; The group-specific primers designed based on exon 4 of the HLA-DRB1 gene include: DRB1 The amplification primer sequences for alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.16 and SEQ ID NO.17; DRB1 The amplification primer sequences for the 04 / 07 / 09 alleles are shown in SEQ ID NO.18 and SEQ ID NO.19; DRB1 The primer sequences for amplification of the 10 alleles are shown in SEQ ID NO.20 and SEQ ID NO.

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3. The application of the primer set according to any one of claims 1-2 in the preparation of HLA-DRB1 gene high-resolution typing products, characterized in that, In the aforementioned application, the method for high-resolution HLA-DRB1 gene typing includes the following steps: (1) Extract genomic DNA from the sample to be tested and dilute it to 10 ng / μl according to the DNA quantification results; (2) The DNA of the test sample was amplified by PCR using the group-specific amplification primers to obtain amplification products containing exons 1, 2, 3 and 4 of the HLA-DRB1 gene, respectively. (3) Perform agarose gel electrophoresis on the amplification products; (4) Sequencing the PCR products using sequencing primers; (5) The sequencing results were compared with the standard exon sequences of the HLA-DRB1 gene in the IMGT / HLA database to finally determine the type of the HLA-DRB1 gene.

4. The application according to claim 3, characterized in that, The amplification system in step (2) is 10 μl, containing 5 μl of 2×Taq Master mix or 2×HotStart Taq Master mix, 0.4 μl of F primer, 0.4 μl of R primer, 0.5 μl of template DNA, and ddH2O added to 10 μl.

5. The application according to claim 4, characterized in that, The 2×Taq Master mix is ​​used to amplify DRB1 in exon 1. 01 / 15 / 16 alleles, DRB1 Alleles 03 / 08 / 10 / 11 / 12 / 13 / 14; DRB1 in exon 2 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 Alleles, DRB1 07 / 09 alleles, DRB1 10 alleles; DRB1 in exon 3 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles; and DRB1 in exon 4 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 alleles, DRB1 04 / 07 / 09 alleles, DRB1 10 alleles; The 2×HotStart Taq Master mix is ​​used to amplify DRB1 in exon 1. 04 / 07 / 09 alleles.

6. The application according to claim 5, characterized in that, In step (2): HLA-DRB1 gene exon 1 DRB1 01 / 15 / 16 alleles and DRB1 The reaction conditions for allele PCR amplification on March 8th, 10th, 11th, 12th, 13th, and 14th were: 94℃, 3 min; 94℃, 20 s → 61℃, 20 s → 72℃, 40 s (35 cycles); 72℃, 5 min; for HLA-DRB1 gene exon 1 DRB1. The reaction conditions for allele PCR amplification on April 7th and September 2009 were as follows: 94℃, 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 63℃, 20 s → 72℃, 40 s (29 cycles); 72℃, 5 min. HLA-DRB1 gene exon 2 DRB1 The reaction conditions for allele PCR amplification on January 1st, 3rd, 4th, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 72℃ for 20 seconds → 72℃ for 40 seconds (10 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 seconds → 67℃ for 20 seconds → 72℃ for 40 seconds (25 cycles); 72℃ for 5 minutes; for exon 2 DRB1 of the HLA-DRB1 gene. The reaction conditions for PCR amplification of the 07 / 09 alleles were: 94℃ for 3 min; 94℃, 20 s → 67℃, 20 s → 72℃, 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 65℃, 20 s → 72℃, 40 s (31 cycles); 72℃ for 5 min; targeting exon 2 DRB1 of the HLA-DRB1 gene. The reaction conditions for PCR amplification of 10 alleles were 94℃ for 3 min; 94℃ for 20 s → 70℃ for 20 s → 72℃ for 30 s (35 cycles); 72℃ for 5 min. HLA-DRB1 gene exon 3 DRB1 The reaction conditions for allele PCR amplification on January 1st, 3rd, 8th, 11th, 12th, 13th, 14th, 15th, and 16th were: 94℃ for 3 minutes; 94℃ for 20 seconds → 63℃ for 20 seconds → 72℃ for 40 seconds (35 cycles); 72℃ for 5 minutes; for exon 3 of the HLA-DRB1 gene, DRB1. The reaction conditions for allele PCR amplification on April 7th, 2009 were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (35 cycles); 72℃ for 5 min; targeting exon 3 of the HLA-DRB1 gene, DRB1. The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (33 cycles); 72℃ for 5 min. HLA-DRB1 gene exon 4 DRB1 The reaction conditions for allele PCR amplification on January 1, 3, 8, 11, 12, 13, 14, 15, and 16 were: 94℃ for 3 min; 94℃ for 20 s → 63℃ for 20 s → 72℃ for 40 s (35 cycles); 72℃ for 5 min; for exon 4 DRB1 of the HLA-DRB1 gene. The reaction conditions for allele PCR amplification on April 7th, 2009 were: 94℃ for 3 min; 94℃, 20 s → 64℃, 20 s → 72℃, 60 s (8 cycles, decreasing by 0.5℃ per cycle); 94℃, 20 s → 60℃, 20 s → 72℃, 60 s (27 cycles); 72℃ for 5 min; targeting exon 4 of the HLA-DRB1 gene, DRB1. The reaction conditions for PCR amplification of 10 alleles were: 94℃ for 3 min; 94℃ for 20 s → 64℃ for 20 s → 72℃ for 40 s (4 cycles, decreasing by 0.5℃ per cycle); 94℃ for 20 s → 62℃ for 20 s → 72℃ for 40 s (31 cycles); 72℃ for 5 min.

7. The application according to claim 6, characterized in that, In step (3), 2% agarose gel is used, the voltage is 120V, and the time is 30min.

8. The application according to claim 7, characterized in that, In step (4): The sequencing primer sequences for all alleles of exon 1 of the HLA-DRB1 gene are shown in SEQ ID NO.1; HLA-DRB1 gene exon 2 DRB1 The sequencing primer sequences for alleles 01 / 03 / 04 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO. 5, DRB1. The sequencing primer sequences for the 07 / 09 alleles are shown in SEQ ID NO.7, DRB1 The sequencing primer sequences for the 10 alleles are shown in SEQ ID NO. 9; HLA-DRB1 gene exon 3 DRB1 The sequencing primer sequences for the alleles 01 / 03 / 08 / 11 / 12 / 13 / 14 / 15 / 16 are shown in SEQ ID NO.11 and SEQ ID NO.12, DRB1 The sequencing primer sequences for the 04 / 07 / 09 alleles are shown in SEQ ID NO. 13, DRB1. The sequencing primer sequences for the 10 alleles are shown in SEQ ID NO.15; The sequencing primer sequences for all alleles of exon 4 of the HLA-DRB1 gene are shown in SEQ ID NO.

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9. A kit for high-resolution HLA-DRB1 gene typing, characterized in that, The kit contains the primer set as described in any one of claims 1-2.

10. The reagent kit according to claim 9, characterized in that, The kit also contains sequencing primers used in any of the applications described in claims 3-8.

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