Primers for detecting full lengths of human HLA-I and HLA-II genes and kit for detecting full lengths of human HLA-I and HLA-II genes

Abstract

The invention discloses a group of primers for detecting full lengths of human HLA-class I and HLA-class II genes and a kit for detecting the full lengths of the human HLA-class I and HLA-class II genes. The group of primers for detecting the full lengths of the human HLA-class I and class II genes comprises primers as shown in SEQ ID No.: 002-SEQ ID No.: 007 in a sequence table. The group of primers can perform multiple PCR amplification in a single tube, target sequences of 11 HLA genes can be amplified at the same time, the HLA amplification cost is reduced, and the operation is simpler and more convenient.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular, to a set of primers for detecting the full-length human HLA class I and HLA class II genes and a kit thereof. Background Art

[0002] Human leukocyte antigen (HLA) typing detection is a medical test used to determine the HLA similarity between individuals, mainly for tissue typing before organ transplantation. HLA typing detection has great market potential. In terms of organ transplantation: The number of people registered in China waiting for organ transplantation has exceeded 140,000, but the number of patients receiving organ transplantation each year is less than 20,000 cases (the ratio of transplant recipients to donors is 1:7), ranking second in the world. Currently, HLA typing detection is mainly applied to the field of kidney transplantation, with a scale of about 150 million yuan. According to the "Consensus Guidelines on HLA-related Detection and Clinical Management Issues in Transplantation", it is recommended that all organ transplantation surgeries perform HLA typing detection. If so, the scale would be 240 million yuan. In terms of hematopoietic stem cell transplantation (HSCT): The number of HSCT cases in China accounts for 14.9% of the total number of global hematopoietic stem cell transplantation cases, and this data is still increasing. In 2021, the number of HSCT cases in China reached 18,218.

[0003] Transplant HLA matching refers to detecting the compatibility of HLA between donors and recipients through serological or molecular biological techniques. The better the HLA compatibility, the lower the probability of rejection after transplantation. Due to the complexity of HLA, it is difficult to find donors and recipients with completely matching types except for identical twins. Therefore, HLA matching is an important test item for donor selection before organ or stem cell transplantation.

[0004] The number of organ transplantation and hematopoietic stem cell transplantation patients in China is increasing. Through HLA typing detection, the extremely precious organ and hematopoietic stem cell resources can be efficiently utilized, and the probability of receptor rejection can be reduced to benefit the patients. Therefore, HLA typing detection has significant clinical value. Summary of the Invention

[0005] An object of the present invention is to provide a set of primers for detecting the full-length human HLA class I and HLA class II genes and a kit thereof.

[0006] A set of primers provided by the present invention for detecting the full lengths of human HLA class I and HLA class II genes, including the primers shown in SEQ ID No.: 002-SEQ ID No.: 007 in the sequence listing, and this primer set is named C3-1. HLA class I gene typing is achieved by using the primer set C3-1 and based on the third-generation sequencing technology platform.

[0007] In one embodiment, the primers further include the primers shown in SEQ ID No.: 008-SEQ ID No.: 017, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 042, SEQ ID No.: 043 in the sequence listing, and this primer set is named C3. HLA class I gene and HLA class II gene typing are achieved by using the primer set C3 and based on the third-generation sequencing technology platform.

[0008] In one embodiment, the primers further include the primers shown in SEQ ID No.: 008-SEQ ID No.: 017, SEQ ID No.: 020, SEQ ID No.: 021, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035 in the sequence listing, and this primer set is named C1. HLA class I gene and HLA class II gene typing are achieved by using the primer set C1 and based on the third-generation sequencing technology platform.

[0009] In one embodiment, the primers further include the primers shown in SEQ ID No.: 008-SEQ ID No.: 013, SEQ ID No.: 016, SEQ ID No.: 017, SEQ ID No.: 020, SEQ ID No.: 021, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 040, SEQ ID No.: 041 in the sequence listing, and this primer set is named C2. HLA class I gene and HLA class II gene typing are achieved by using the primer set C2 and based on the third-generation sequencing technology platform.

[0010] In one embodiment, the primers further include the primers shown in SEQ ID No.:008, SEQ ID No.:009, SEQ ID No.:012, SEQ ID No.:013, SEQ ID No.:016, SEQ ID No.:017, SEQ ID No.:020, SEQ ID No.:021, SEQ ID No.:026, SEQ ID No.:027, SEQ ID No.:034, SEQ ID No.:035, SEQ ID No.:040, SEQ ID No.:041, SEQ ID No.:044, SEQ ID No.:045 in the sequence listing. This primer set is named C4. HLA class I gene and HLA class II gene typing were achieved by using primer set C4 and based on the third-generation sequencing technology platform.

[0011] Another object of the present invention is to provide a kit for detecting HLA class I and HLA class II genes.

[0012] The kit provided by the present invention contains the above primer sets C3-1, C3, C1, C2 or C4.

[0013] Another object of the present invention is to provide a method for HLA gene typing based on the third-generation sequencing technology platform.

[0014] The method for HLA gene typing based on the third-generation sequencing technology platform provided by the present invention includes: Performing PCR amplification on the sample to be tested using the above primer sets C3-1, C3, C1, C2 or C4 to obtain an amplification product; Constructing an HLA gene sequencing library based on the third-generation sequencing technology platform using the amplification product; Sequencing the HLA gene sequencing library on the third-generation sequencing technology platform and performing typing on the sequencing data.

[0015] In one embodiment, constructing the HLA gene sequencing library based on the third-generation sequencing technology platform using the amplification product includes: Performing end modification on the amplification product; Making the end-modified amplification product ligate with a linker barcode to obtain a ligation product; Making the ligation product ligate with a sequencing adapter and performing library amplification to obtain the HLA gene sequencing library.

[0016] Another object of the present invention is to provide a kit for HLA gene typing based on the third-generation sequencing technology platform.

[0017] The HLA genotyping kit provided by the present invention comprises primer sets C3-1, C3, C1, C2 or C4.

[0018] In one embodiment, the HLA genotyping kit further comprises a connection barcode, a sequencing adapter, a terminal modification enzyme, and a ligase for constructing an HLA gene sequencing library.

[0019] The present invention provides a set of primers for detecting HLA-I and HLA-II genes, which can perform multiple PCR amplification in a single tube, and can simultaneously amplify the target sequences of 11 HLA genes, thereby reducing the cost of HLA amplification and making the operation simpler, thereby significantly improving the detection turnaround time, reducing costs and shortening the total reaction time. The present invention provides a set of primers for detecting HLA-I and HLA-II genes, combined with a third-generation sequencing technology platform, can simultaneously detect 11 HLA genes and perform HLA genotyping on them. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis diagram of the HLA polymorphism primers of Example 1 of the present invention is shown; Figure 2 The specific electrophoresis diagram of the HLA polymorphism single primer amplification of Example 2 of the present invention is shown; Figure 3 The specific electrophoresis diagram of the HLA polymorphism primer set amplification in Example 3 of the present invention is shown; Figure 4 A schematic diagram of the HLA third-generation sequencing amplicon technology in Example 4 of the present invention is shown; Figure 5 The electrophoresis diagram of the HLA third generation sequencing amplicon in Example 4 of the present invention is shown; Figure 6 The quality control diagram of HLA third generation sequencing in Example 4 of the present invention is shown; Figure 7 The data splitting and yield diagram of HLA third generation sequencing in Example 4 of the present invention are shown; Figure 8 The HLA third generation sequencing data typing results in Example 4 of the present invention are shown; Figure 9A , Figure 9B and Figure 9C A comparison diagram of the HLA third-generation sequencing data typing site verification in Example 4 of the present invention is shown; Figure 10 The electrophoresis diagram of the HLA third generation sequencing amplicon in Example 5 of the present invention is shown; Figure 11 The data splitting and yield diagram of HLA third generation sequencing in Example 5 of the present invention are shown; Figure 12The HLA third-generation sequencing data typing results in Embodiment 5 of the present invention are shown. Detailed implementation manners

[0021] To make the technical solutions of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Embodiment 1

[0023] Verification of HLA polymorphism primers:

[0024] In this embodiment, the amplification performance and amplification specificity of a conventional primer pair and a polymorphism primer pair were compared.

[0025] Compared with the conventional primer SEQ ID No.: 001 and the polymorphism primer SEQ ID No.: 003, the nucleotide at the fourth position is different. The conventional primer is a and the polymorphism primer is r. Where r represents a / g.

[0026] Amplification: Pipette 10 ng of DNA from a blood sample, prepare an amplification system based on Table 1. Add the conventional primer pair (the sequences of the forward primer SEQ ID No.: 001 and the reverse primer SEQ ID No.: 002 are shown in Table 2) to tube A1 in the amplification system, and add the polymorphism primer pair (the forward primer SEQ ID No.: 002 and the reverse primer SEQ ID No.: 003) to tube A2 in the amplification system. Both are made up to 50 μL with water, run PCR, 95°C, 3 min; (95°C, 15 s; 58°C, 15 s; 72°C, 10 min; 35 cycles); 72°C, 5 min; 4°C, hold.

[0027] Purification: Add 0.8× volume of DNA magnetic beads, place at room temperature for 10 min, then place on a magnetic rack for 5 min to adsorb, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 21 μL of water for elution, and take 1 μL of the product for quantification. The product obtained with the conventional primer pair was 0.72 ng / μL, and the product obtained with the polymorphism primer pair was 93.4 ng / μL.

[0028] Quantification and electrophoresis: Take 50 ng of the purified product for agarose electrophoresis to identify primer specificity. The electrophoresis pattern is as Figure 1 shown, A1 is the conventional primer pair, and A2 is the polymorphism primer pair.

[0029] The results show that the polymorphism primer pair has better amplification performance and amplification specificity compared with the conventional primer pair. This also shows the particularity of the HLA gene. SNPs in the HLA gene located in the region where the primer binds to the template may lead to a decrease in the binding efficiency of the primer to the template DNA, greatly inhibiting the PCR reaction, and at the same time may cause the primer to fail to bind to the target region, thus amplifying non-specific products.

[0030] Example 2 HLA single primer screening:

[0031] In this example, primer pairs targeting different HLA genes were designed, and the amplification performance and amplification specificity of different primer pairs were compared.

[0032] Amplification: Pipette 10 ng of DNA from the blood sample, prepare the amplification system based on Table 1, add primer pairs 1 - 19 to tubes B1 - B19 respectively (primer pairs 1 - 19 are shown in Table 3, and the primer sequences are shown in Table 2), make up to 50 µL with water, run PCR, 95°C, 3 min; (95°C, 15 s; 58°C, 15 s; 72°C, 10 min; 35 cycles); 72°C, 5 min; 4°C, hold.

[0033] Purification: Add 0.8× volume of DNA magnetic beads, place at room temperature for 10 min, then place on the magnetic rack for 5 min to adsorb, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 21 µL of water for elution, and take 1 µL of the product for quantification. The quantification results show that the products are all above 50 ng / µL.

[0034] Quantification and electrophoresis: Take 50 ng of the purified product for agarose electrophoresis to identify the primer pair specificity. The corresponding relationship between the primer pairs and the lanes is shown in Table 3, and the electrophoresis results are as Figure 2 shown.

[0035] The electrophoresis results show that there are non - specific fragments and the product amount is low in lanes B9 and B11. Lanes B13 and B17 are relatively better. This indicates that there are significant differences in the amplification specificity of different primer pairs. It can be seen from the electrophoresis results that after multiple rounds of primer pair optimization and screening, stable large - fragment PCR products appear, indicating that single - gene full - length amplification is achieved through the amplification of a single primer pair. After screening, the amplification performance and amplification specificity of primer pairs 1 - 8, primer pair 10, primer pair 13, and primer pair 17 are relatively good.

[0036] Table 1. Amplification reagent system

[0037] Table 2. Primer sequences

[0038] Table 3. Corresponding relationship between primer pairs, genes, and lanes

[0039] Example 3 Amplification efficiency and specificity of HLA primer sets:

[0040] In this embodiment, different HLA primer sets were designed, and primer sets with high amplification efficiency and specificity were obtained through screening to reduce costs and improve operational convenience.

[0041] Amplification: Pipette 10 ng of DNA from the blood sample, prepare the amplification system based on Table 1, add the corresponding primer sets (the primer sets are shown in Table 4), make up to 50 µL with water, run PCR, 95 °C, 3 min; (95 °C, 15 s; 58 °C, 15 s; 72 °C, 10 min; 35 cycles); 72 °C, 5 min; 4 °C, hold.

[0042] Purification: Add 0.8× volume of DNA magnetic beads, place at room temperature for 10 min, then place on a magnetic stand to adsorb for 5 min, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 21 µL of water for elution, and take 1 µL of the product for quantification. The total amount of the product is 2000 ng.

[0043] Quantification and electrophoresis: Take 50 ng of the purified product for agarose electrophoresis to identify the amplification efficiency and specificity of the primer sets. The results are as Figure 3 .

[0044] Table 4. Composition of primer sets and corresponding lanes

[0045] The electrophoresis results showed that lanes C1, C2, C3, and C4 were better than lane C5. Lane C3 was the best, with obvious main peaks from 3000 bp to 12000 bp and fewer dimers. The PCR product in lane C3 reached 1.5 µg with a 10 ng template. This indicates that primer sets C1, C2, C3, and C4 had better amplification efficiency and specificity compared to primer set C5. Primer set C3 had the best amplification efficiency and specificity. Through the screened primer sets C1, C2, C3, and C4, this embodiment achieved the function of amplifying 11 HLA genes in a single tube, reduced the cost of conventional HLA amplification, made the operation more convenient, and shortened the total reaction time while reducing costs.

[0046] Example 4 Accuracy of HLA-I type in clinical samples:

[0047] To further illustrate the flexibility of the primer sets of the present invention, in this embodiment, by using primer set C3-1 (SEQ ID No.: 002 - SEQ ID No.: 007) and based on the third-generation sequencing technology platform (abbreviated as HLA third-generation sequencing), HLA class I gene typing was achieved ( Figure 4 ).

[0048] Extract the DNA from the blood sample and perform quality control with Qubit.

[0049] Amplification: Pipette 10 ng of DNA, prepare the amplification system based on Table 1, use Primer Set C3-1 for the primer set, make up to 50 µL with water, run PCR, 95°C, 3 min; (95°C, 15 s; 58°C, 15 s; 72°C, 10 min; 35 cycles); 72°C, 5 min; 4°C, hold. After amplification is completed, it can be stored at -20°C for 1 month; Purification: Add 50 µL of DNA magnetic beads to the above reaction system. After standing at room temperature for 10 min, place it on a magnetic stand to adsorb for 5 min, then discard the supernatant. Wash twice with 80% ethanol, dry at room temperature for 5 min, elute with 47 µL of water, and take 1 µL of the product for quantification; End repair and addition of "A": Take 45 µL of the above reaction system (Table 5), add 15 µL of the end repair and addition of "A" reaction solution, run PCR, 20°C, 10 min; 65°C, 10 min; 4°C, hold.

[0050] Purification: Add 60 µL of DNA magnetic beads to the above reaction system. After standing at room temperature for 10 min, place it on a magnetic stand to adsorb for 5 min, then discard the supernatant. Wash twice with 80% ethanol, dry at room temperature for 5 min, elute with 23 µL of water, and take 1 µL of the product for quantification; Ligation of Index: Take 21 µL of the above reaction system (Table 5), add 5 µL of the ligation barcode BL01 to the above reaction system, add 14 µL of Ligation Reaction Solution 1, run PCR, 25°C, 30 min; Purification: Add 2 µL of EDTA to the above reaction system, add 0.4× volume of DNA magnetic beads. After standing at room temperature for 10 min, place it on a magnetic stand to adsorb for 5 min, then discard the supernatant. Wash twice with 80% ethanol, dry at room temperature for 5 min, elute with 32 µL of water, and take 1 µL for quantification; Ligation of adapter: Add 5 µL of the sequencing adapter and 70 µL of Ligation Reaction Solution 2 to the above reaction system, run PCR, 25°C, 30 min; Purification: Add 80 µL of DNA magnetic beads to the above reaction system. After standing at room temperature for 10 min, place it on a magnetic stand to adsorb for 5 min, then discard the supernatant. Wash twice with 300 µL of the long fragment cleaning solution, elute with 42 µL of water, and take 1 µL for quantification. The total amount of the product is greater than 2000 ng, take 50 ng for electrophoresis. The electrophoresis result shows three bands corresponding to HLA-A, HLA-B, and HLA-C, as Figure 5 shown.

[0051] Sequencing and data analysis: Merge the libraries according to the BGISEQ-500 third-generation on-machine process. The amount of data downloaded is more than 1 G, meeting the requirements. Carry out analysis according to the Human HLA Third-generation Sequencing Analysis Process V1.

[0052] Table 5. Materials used in Example 4

[0053] The use of primer set C3-1 can achieve the amplification of HLA class I genes, and the data quality control values are all above 8 ( Figure 6 ). After Index splitting, the target data volume is above 1G, meeting the expectations and analysis requirements ( Figure 7 ).

[0054] Compared with the comparative example (detecting SNPs by PCR method), for the genotyping results using primer set C3-1, the results of HLA-B / C are completely consistent with those of the comparative example, and there is one allele of HLA-A that is inconsistent. Among them, for the genotyping of HLA-A*07:02 in the comparative example, the corresponding supporting reads were also found in the HLA third-generation sequencing off-machine data, but the number of supports is very small; there are more reads supporting HLA-A*11:413N (as Figure 8 shown).

[0055] Furthermore, for the above two genotypings of the HLA-A gene, a check was carried out. The checking idea was to search for the sequences of the two genotypings in the IMGT database. The checking results found that the main difference between the above two genotypings was in the Exon2 and Exon3 regions.

[0056] The SNP difference sites of the two genotypings in the above two regions were rechecked. The recheck results are as Figure 9A , Figure 9B and Figure 9C shown. The bases of the HLA third-generation sequencing off-machine data are completely matched with HLA-A*11:413N. This indicates that due to the advantage of the read length of HLA third-generation sequencing, all SNPs on the gene can be included to assist in genotyping, and the advantage of HLA third-generation sequencing is obvious. Using the PCR method to detect SNPs for HLA gene genotyping can only be based on a few SNPs for genotyping, and requires more primer sets, making it difficult to cover more genotyping types; and using the PCR method to detect SNPs cannot identify the abundance differences of different genotypings, select the genotyping with the highest number of supports, and also cannot include multiple sites for statistics to assist in genotyping. Comparing the genotyping sequence with the highest number of supports in the original HLA third-generation sequencing off-machine data extracted with the sequence of this genotyping in the IMGT database, it was found that the genotyping sequence with the highest number of supports was completely consistent with that in the database and was very different from the sequence of the comparative example.

[0057] Example 5 Accuracy of HLA-II type in clinical samples:

[0058] To further illustrate the flexibility and accuracy of the primer set of the present invention, in this example, HLA class II genotyping was achieved by using primer set C3 (SEQ ID No.: 002 - SEQ ID No.: 017, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 042, SEQ ID No.: 043) and based on the third-generation sequencing technology platform (abbreviated as HLA third-generation sequencing).

[0059] Extract DNA from the blood sample and perform quality control with Qubit.

[0060] Amplification: Pipette 10 ng of DNA, prepare the amplification system based on Table 1, use primer set C3 as the primer set, make up to 50 µL with water, run PCR, 95°C, 3 min; (95°C, 15 s; 58°C, 15 s; 72°C, 10 min; 35 cycles); 72°C, 5 min; 4°C, hold. After amplification is completed, it can be stored at -20°C for 1 month; Purification: Add 50 µL of DNA magnetic beads to the above reaction system, place it at room temperature for 10 min, then place it on a magnetic rack to adsorb for 5 min, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 47 µL of water for elution, and take 1 µL of the product for quantification; End repair and addition of "A": Take 45 µL of the above reaction system (Table 5), add 15 µL of the end repair and addition of "A" reaction solution, run PCR, 20°C, 10 min; 65°C, 10 min; 4°C, hold.

[0061] Purification: Add 60 µL of DNA magnetic beads to the above reaction system, place it at room temperature for 10 min, then place it on a magnetic rack to adsorb for 5 min, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 23 µL of water for elution, and take 1 µL of the product for quantification; Ligation of Index: Take 21 µL of the above reaction system (Table 5), add 5 µL of the ligation barcode BL01 to the above reaction system, add 14 µL of ligation reaction solution 1, run PCR, 25°C, 30 min; Purification: Add 2 µL of EDTA to the above reaction system, add 0.4× volume of DNA magnetic beads, place it at room temperature for 10 min, then place it on a magnetic rack to adsorb for 5 min, discard the supernatant, wash twice with 80% ethanol, air dry at room temperature for 5 min, add 32 µL of water for elution, and take 1 µL for quantification; Ligation of adapter: Add 5 µL of the sequencing adapter and 70 µL of ligation reaction solution 2 (Table 5) to the above reaction system, run PCR, 25°C, 30 min; Purification: Add 80 µL of DNA magnetic beads to the above reaction system. After 10 min at room temperature, place it on a magnetic stand and adsorb for 5 min, then discard the supernatant. Wash twice with 300 µL of long fragment washing solution, and then elute with 42 µL of water. Take 1 µL for quantification. If the total amount of the product is greater than 1500 ng, take 50 ng for electrophoresis. The electrophoresis result shows bands corresponding to 3000 bp - 12000 bp ( Figure 10 ).

[0062] Sequencing and data analysis: Merge the libraries according to the BGI third-generation sequencing process. The data volume after sequencing is about 0.2 G, which meets the requirements. Conduct analysis according to the human HLA third-generation sequencing analysis process V1.

[0063] The primer set C3 can be used to amplify HLA class II genes. After Index splitting, the target data volume is about 0.2 G, meeting the expectations and analysis requirements ( Figure 11 ).

[0064] Compared with the comparative example (detecting SNPs by PCR method), for the genotyping results using the primer set C3, the DRB1 and DQB1 genotyping of HLA-II are completely consistent with the results of the comparative example, and the number of supporting reads is more than 300 for both ( Figure 12 ).

[0065] The results of this example show that the primer set C3 can also amplify HLA class II genes well. Moreover, the present invention is flexible in use, can adapt to the detection of HLA class II genes, meet different requirements, and is easy to operate.

[0066] The above-described embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A set of primers for detecting HLA class I and HLA class II genes, characterized in that, The primers include the primers shown in SEQ ID No.: 002 - SEQ ID No.: 007 in the sequence listing.

2. The primer according to claim 1, characterized in that, The primers further include the primers shown in SEQ ID No.: 008 - SEQ ID No.: 017, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 042, SEQ ID No.: 043 in the sequence listing.

3. The primer according to claim 1, characterized in that, The primers further include the primers shown in SEQ ID No.: 008 - SEQ ID No.: 017, SEQ ID No.: 020, SEQ ID No.: 021, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035 in the sequence listing.

4. The primer according to claim 1, wherein The primers further include the primers shown in SEQ ID No.: 008 - SEQ ID No.: 013, SEQ ID No.: 016, SEQ ID No.: 017, SEQ ID No.: 020, SEQ ID No.: 021, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 040, SEQ ID No.: 041 in the sequence listing.

5. The primer according to claim 1, characterized in that, The primers further include the primers shown in SEQ ID No.: 008, SEQ ID No.: 009, SEQ ID No.: 012, SEQ ID No.: 013, SEQ ID No.: 016, SEQ ID No.: 017, SEQ ID No.: 020, SEQ ID No.: 021, SEQ ID No.: 026, SEQ ID No.: 027, SEQ ID No.: 034, SEQ ID No.: 035, SEQ ID No.: 040, SEQ ID No.: 041, SEQ ID No.: 044, SEQ ID No.: 045 in the sequence listing.

6. Use of the primers according to any one of claims 1 to 5 in detecting HLA class I and HLA class II genes.

7. A kit for detecting HLA class I and HLA class II genes, characterized in that, The kit contains the primers according to any one of claims 1 to 5.

8. An HLA genotyping method based on a third-generation sequencing technology platform, comprising: Performing PCR amplification on a sample to be tested using the primers according to any one of claims 1 to 5 to obtain an amplification product; Constructing an HLA gene sequencing library based on the third-generation sequencing technology platform using the amplification product; Sequencing the HLA gene sequencing library on the third-generation sequencing technology platform and genotyping the sequencing data.

9. The HLA gene typing method according to claim 8, characterized in that, Constructing an HLA gene sequencing library based on the third-generation sequencing technology platform using the amplification product includes: Performing end modification on the amplification product; Connecting the end-modified amplification product with a linker barcode to obtain a ligation product; Connecting the ligation product with a sequencing adapter and performing library amplification to obtain the HLA gene sequencing library.

10. An HLA gene typing kit based on the third-generation sequencing technology platform, comprising the primer according to any one of claims 1 to 5.

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