A set of primers and kits for detecting the full length of human HLA-I and HLA-II genes

Abstract

The present invention discloses a set of primers and a kit for detecting the full-length human HLA class I and class II genes. This set of primers includes primers shown in the sequence listing as SEQ ID No. 002 and SEQ ID No. 007. This primer set enables multiplex PCR amplification in a single tube, simultaneously amplifying target sequences of 11 HLA genes, reducing the cost of HLA amplification and simplifying the procedure.

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 of human HLA-I and HLA-II genes and a kit thereof. Background Art

[0002] Human leukocyte antigen (HLA) typing is a medical test used to determine HLA similarity between individuals. It is primarily used for tissue matching prior to organ transplantation. HLA typing has enormous market potential. In organ transplantation, my country has over 140,000 registered candidates for organ transplants, but fewer than 20,000 patients receive organ transplants annually (with a recipient-to-donor ratio of 1:7), ranking second worldwide. Currently, HLA typing is primarily used in kidney transplantation, with a total investment of approximately 150 million yuan. The "Consensus Guidelines on HLA-Related Testing and Clinical Management in Transplantation" recommend that all organ transplants undergo HLA typing, a cost of 240 million yuan. Regarding hematopoietic stem cell transplantation (HSCT), my country accounts for 14.9% of the global total, and this figure continues to grow. In 2021, 18,218 HSCTs were performed in my country.

[0003] HLA matching for transplantation refers to the testing of HLA compatibility between the donor and recipient using serological or molecular biology techniques. The better the HLA compatibility, the lower the chance of rejection after the transplant. Due to the complexity of HLA, finding a perfectly matched donor and recipient is difficult, except for identical twins. Therefore, HLA matching is a key test for donor selection before organ or stem cell transplantation.

[0004] The number of patients undergoing organ transplantation and hematopoietic stem cell transplantation in my country is increasing. HLA typing testing can not only efficiently utilize extremely precious organs and hematopoietic stem cell resources, but also reduce the chance of recipient rejection reaction to benefit patients. Therefore, HLA typing testing has significant clinical value. Summary of the Invention

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

[0006] The present invention provides a set of primers for detecting the full-length human HLA-I and HLA-II genes, including primers shown in the sequence listing as SEQ ID No.: 002 to SEQ ID No.: 007. This primer set is designated C3-1. Using primer set C3-1, HLA-I genotyping is achieved using a third-generation sequencing platform.

[0007] In one embodiment, the primers further include primers set forth 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, and SEQ ID No.: 043 in the sequence listing, and this primer set is designated C3. HLA class I and HLA class II genotyping is achieved using primer set C3 and a third-generation sequencing technology platform.

[0008] In one embodiment, the primers further include primers set forth 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, and SEQ ID No.: 035 in the sequence listing, and this primer set is designated C1. HLA class I and HLA class II genotyping is achieved using primer set C1 and a third-generation sequencing technology platform.

[0009] In one embodiment, the primers further include primers set forth 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, and SEQ ID No.: 041 in the sequence listing, and this primer set is designated C2. HLA class I and HLA class II genotyping is achieved using primer set C2 and a third-generation sequencing technology platform.

[0010] In one embodiment, the primers further include primers set forth 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, and SEQ ID No.: 045 in the sequence listing, and this primer set is designated C4. HLA class I and HLA class II genotyping is achieved using primer set C4 and a third-generation sequencing technology platform.

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

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

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

[0014] The HLA genotyping method based on the third-generation sequencing technology platform provided by the present invention includes:

[0015] Perform PCR amplification on the sample to be tested using the primer set C3-1, C3, C1, C2 or C4 to obtain an amplified product;

[0016] Using the amplified product to construct an HLA gene sequencing library based on a third-generation sequencing technology platform;

[0017] The HLA gene sequencing library is sequenced on a third-generation sequencing technology platform and the sequencing data is typed.

[0018] In one embodiment, the use of the amplified products to construct an HLA gene sequencing library based on a third-generation sequencing technology platform comprises:

[0019] performing end modification on the amplified product;

[0020] connecting the terminally modified amplified product to the connection barcode to obtain a connection product;

[0021] The ligation product is connected to a sequencing adapter, and library amplification is performed to obtain the HLA gene sequencing library.

[0022] Another object of the present invention is to provide an HLA genotyping kit based on a third-generation sequencing technology platform.

[0023] The HLA genotyping kit provided by the present invention contains primer sets C3-1, C3, C1, C2 or C4.

[0024] 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.

[0025] The present invention's set of primers for detecting HLA-I and HLA-II genes enables multiplex PCR amplification in a single tube, simultaneously amplifying target sequences for 11 HLA genes. This reduces the cost of HLA amplification and simplifies the process, significantly improving test turnaround time. This reduces costs while shortening overall reaction time. Combined with a third-generation sequencing platform, this set of primers for detecting HLA-I and HLA-II genes can simultaneously detect and perform HLA genotyping on 11 HLA genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows the electrophoresis diagram of the HLA polymorphism primers of Example 1 of the present invention;

[0027] Figure 2 The figure shows the specific electrophoresis diagram of the HLA polymorphism single primer amplification in Example 2 of the present invention;

[0028] Figure 3 shows the specific electrophoresis diagram of the HLA polymorphism primer set amplification in Example 3 of the present invention;

[0029] Figure 4 A schematic diagram of the HLA third-generation sequencing amplicon technology in Example 4 of the present invention is shown;

[0030] Figure 5 The electrophoresis diagram of the HLA third-generation sequencing amplicon in Example 4 of the present invention is shown;

[0031] Figure 6 The figure shows the quality control chart of HLA third-generation sequencing in Example 4 of the present invention;

[0032] Figure 7 The figure shows the splitting and yield of HLA third-generation sequencing data in Example 4 of the present invention;

[0033] Figure 8 The HLA third-generation sequencing data typing results in Example 4 of the present invention are shown;

[0034] 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;

[0035] Figure 10 The figure shows the electrophoresis diagram of HLA third generation sequencing amplicon in Example 5 of the present invention;

[0036] Figure 11 The figure shows the splitting and yield of HLA third-generation sequencing data in Example 5 of the present invention;

[0037] Figure 12 The data show the typing results of HLA third-generation sequencing data in Example 5 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] HLA polymorphism primer verification:

[0041] This example compares the amplification performance and amplification specificity of conventional primer pairs and polymorphic primer pairs.

[0042] The conventional primer SEQ ID No.: 001 and the polymorphic primer SEQ ID No.: 003 differ in the fourth nucleotide position, the conventional primer being a and the polymorphic primer being r, wherein r represents a / g.

[0043] Amplification: Pipette 10 ng of DNA from a blood sample and prepare an amplification system based on Table 1. Add a conventional primer pair (forward primer SEQ ID No.: 001 and rear primer SEQ ID No.: 002, as shown in Table 2) to tube A1, and a polymorphic primer pair (forward primer SEQ ID No.: 002 and rear primer SEQ ID No.: 003) to tube A2. Make up the volume to 50 µL with water. Run PCR at 95°C for 3 min, followed by 35 cycles of 95°C for 15 sec, 58°C for 15 sec, and 72°C for 10 min, followed by 72°C for 5 min and a 4°C hold.

[0044] Purification: Add 0.8x the volume of DNA magnetic beads and allow to adsorb on a magnetic rack for 5 minutes at room temperature for 10 minutes. Discard the supernatant, rinse twice with 80% ethanol, and air-dry at room temperature for 5 minutes. Elute with 21 µL of water and quantify 1 µL of product. The product obtained with the conventional primer pair was 0.72 ng / µL, and the product obtained with the polymorphic primer pair was 93.4 ng / µL.

[0045] Quantification and electrophoresis: Take 50ng of purified product and perform agarose electrophoresis to identify primer specificity. Figure 1 As shown, A1 is a conventional primer pair and A2 is a polymorphic primer pair.

[0046] The results showed that polymorphic primer pairs had better amplification performance and specificity than conventional primer pairs. This also reflects the specificity of the HLA gene. SNPs in the HLA gene located in the primer-template binding region may reduce the binding efficiency of the primer and template DNA, greatly inhibiting the PCR reaction. At the same time, it may also cause the primer to fail to bind to the target region, thereby amplifying non-specific products.

[0047] Example 2

[0048] HLA single primer screening:

[0049] In this example, primer pairs targeting different HLA genes were designed, and the amplification performance and amplification specificity of the different primer pairs were compared.

[0050] Amplification: Pipette 10 ng of DNA from a blood sample and prepare the amplification system according to Table 1. Add primer pairs 1-19 to tubes B1-B19, respectively (primer pairs 1-19 are shown in Table 3, and primer sequences are shown in Table 2). Make up to 50 µL with water and run the PCR reaction at 95°C for 3 min; (35 cycles of 95°C for 15 sec; 58°C for 15 sec; and 72°C for 10 min); 72°C for 5 min; and 4°C hold.

[0051] Purification: Add 0.8x the volume of DNA magnetic beads, incubate at room temperature for 10 minutes, place on a magnetic stand for 5 minutes, and discard the supernatant. Rinse twice with 80% ethanol and air dry at room temperature for 5 minutes. Elute with 21µL of water and quantify 1µL of the product. Quantification results showed that the product concentration was above 50ng / µL.

[0052] Quantitation and electrophoresis:

[0053] 50 ng of the purified product was subjected to agarose electrophoresis to identify the specificity of the primer pairs. The corresponding relationship between the primer pairs and the lanes is shown in Table 3. The electrophoresis results are shown in Figure 2 shown.

[0054] Electrophoresis results showed that lanes B9 and B11 contained nonspecific fragments and low product amounts. Lanes B13 and B17 performed relatively well. This suggests that the amplification specificity of different primer pairs varies significantly. The electrophoresis results indicate that after multiple rounds of primer pair optimization and screening, stable large-fragment PCR products emerged, demonstrating that full-length amplification of a single gene was achieved using a single primer pair. After screening, primer pairs 1-8, primer pair 10, primer pair 13, and primer pair 17 demonstrated excellent amplification performance and specificity.

[0055] Table 1. Amplification reagent system

[0056]

[0057] Table 2. Primer sequences

[0058]

[0059] Table 3. Correspondence between primer pairs, genes, and lanes

[0060]

[0061] Example 3

[0062] Amplification efficiency and specificity of HLA primer set:

[0063] In this embodiment, different HLA primer sets were designed, and primer sets with high amplification efficiency and specificity were obtained through screening to achieve cost reduction and improve operational convenience.

[0064] Amplification: Pipette 10 ng of DNA from a blood sample and prepare the amplification system according to Table 1. Add the corresponding primer sets (primer sets are shown in Table 4), make up to 50 µL with water, and run the PCR reaction at 95°C for 3 min; (35 cycles of 95°C for 15 sec; 58°C for 15 sec; and 72°C for 10 min); 72°C for 5 min; and hold at 4°C.

[0065] Purification: Add 0.8x volume of DNA magnetic beads, incubate at room temperature for 10 minutes, place on a magnetic rack for 5 minutes, discard the supernatant, rinse twice with 80% ethanol, air dry at room temperature for 5 minutes, elute with 21µL of water, and quantify 1µL of product. The total product amount is 2000ng.

[0066] Quantification and electrophoresis: 50 ng of the purified product was subjected to agarose electrophoresis to identify the amplification efficiency and specificity of the primer set. The results are as follows: Figure 3 .

[0067] Table 4. Primer set composition and corresponding lanes

[0068]

[0069] The electrophoresis results showed that lanes C1, C2, C3, and C4 performed better than lane C5. Lane C3 was the best, with a clear main peak from 3000bp to 12000bp and fewer dimers. The PCR product in lane C3 reached 1.5µg with a template of 10ng. This indicates that primer sets C1, C2, C3, and C4 had better amplification efficiency and specificity than primer set C5. Primer set C3 had the best amplification efficiency and specificity. By screening primer sets C1, C2, C3, and C4, this embodiment achieved the function of amplifying 11 HLA genes in a single tube, reducing the cost of conventional HLA amplification, making the operation simpler, and shortening the total reaction time while reducing costs.

[0070] Example 4

[0071] HLA-I type accuracy of clinical samples:

[0072] To further illustrate the flexibility of the primer set of the present invention, this example uses primer set C3-1 (SEQ ID No.: 002-SEQ ID No.: 007) and a third-generation sequencing technology platform (hereinafter referred to as HLA third-generation sequencing) to achieve HLA-I genotyping ( Figure 4 ).

[0073] DNA extracted from blood samples was quality controlled using Qubit.

[0074] Amplification: Pipette 10 ng of DNA and prepare the amplification system according to Table 1. Use primer set C3-1, make up to 50 µL with water, and run PCR at 95°C for 3 min; (35 cycles of 95°C for 15 sec; 58°C for 15 sec; 72°C for 10 min); 72°C for 5 min; and hold at 4°C. After amplification, store at -20°C for 1 month.

[0075] Purification: Add 50 µL of DNA magnetic beads to the above reaction system, let it stand at room temperature for 10 minutes, place it on a magnetic stand for adsorption for 5 minutes, discard the supernatant, wash it twice with 80% ethanol, dry it at room temperature for 5 minutes, elute it with 47 µL of water, and take 1 µL of the product for quantification;

[0076] End Repair Plus "A": Take 45 µL of the above reaction system (Table 5), add 15 µL of End Repair Plus "A" reaction solution, and run PCR at 20°C for 10 min, 65°C for 10 min, and 4°C for hold.

[0077] Purification: Add 60µL DNA magnetic beads to the above reaction system, incubate at room temperature for 10 minutes, place on a magnetic stand for 5 minutes, discard the supernatant, wash twice with 80% ethanol, dry at room temperature for 5 minutes, elute with 23µL water, and take 1µL of the product for quantification;

[0078] Ligation Index: Take 21 µL of the above reaction system (Table 5), add 5 µL of ligation barcode BL01 to the above reaction system, add 14 µL of ligation reaction solution 1, and run PCR at 25°C for 30 min.

[0079] Purification: Add 2 µL of EDTA to the above reaction system and add 0.4× volume of DNA magnetic beads. After 10 min at room temperature, place on a magnetic stand for 5 min and discard the supernatant. Wash twice with 80% ethanol and dry at room temperature for 5 min. Elute with 32 µL of water and take 1 µL for quantification.

[0080] Adapter ligation: Add 5 µL sequencing adapters and 70 µL ligation buffer 2 to the above reaction system and run PCR at 25°C for 30 min.

[0081] Purification: Add 80µL of DNA magnetic beads to the above reaction system, incubate at room temperature for 10 minutes, place on a magnetic rack for 5 minutes, and discard the supernatant. Wash twice with 300µL of long fragment cleaning buffer, then elute with 42µL of water. Take 1µL for quantification. If the total amount of product is greater than 2000ng, take 50ng for electrophoresis. The electrophoresis results show three bands corresponding to HLA-A, HLA-B, and HLA-C, such as Figure 5 shown.

[0082] Sequencing and data analysis: The libraries were merged according to the BGI third-generation sequencing process. The data volume was more than 1G, which met the requirements. The analysis was carried out according to the human HLA third-generation sequencing analysis process V1.

[0083] Table 5. Materials used in Example 4

[0084]

[0085] Primer set C3-1 can be used to amplify HLA-I genes, and the data quality control values ​​are all above 8 ( Figure 6 ), after the Index is split, the target data volume is more than 1G, which meets the expectations and analysis requirements ( Figure 7 ).

[0086] Compared with the control group (PCR method for SNP detection), the typing results of primer set C3-1 were completely consistent with the control group results for HLA-B / C, and one HLA-A allele was inconsistent. Among them, the typing HLA-A*07:02 of the control group also found corresponding supporting reads in the HLA third-generation sequencing data, but the number of supporting reads was very small; there were more reads supporting HLA-A*11:413N (such as Figure 8 shown).

[0087] Further investigation was conducted on the two HLA-A gene types mentioned above, by searching for sequences of the two types in the IMGT database. The results showed that the differences between the two types were mainly in the Exon2 and Exon3 regions.

[0088] The SNP difference sites of the two types in the above two regions were reviewed. The review results are as follows Figure 9A 、 Figure 9B and Figure 9C As shown, the bases in the HLA third-generation sequencing data are a perfect match for HLA-A*11:413N. This demonstrates that HLA third-generation sequencing, due to its read length advantage, can incorporate all SNPs on the gene to assist in typing, a significant advantage. Using PCR to detect SNPs for HLA genotyping can only be performed based on a small number of SNPs and requires a large number of primer sets, making it difficult to cover a wide range of typing types. Furthermore, using PCR to detect SNPs cannot identify differences in abundance between different typing types, and only selects the type with the highest support. It also cannot include statistics for multiple loci to assist in typing. Comparing the highest-supported typing sequence in the extracted raw HLA third-generation sequencing data with the sequence of that typing in the IMGT database revealed that the highest-supported typing sequence was completely consistent with the database and significantly different from the comparison sequence.

[0089] Example 5

[0090] HLA-II type accuracy of clinical samples:

[0091] To further illustrate the flexibility and accuracy of the primer sets of the present invention, this example utilizes 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 implements HLA class II genotyping based on a third-generation sequencing technology platform (referred to as HLA third-generation sequencing).

[0092] DNA extracted from blood samples was quality controlled using Qubit.

[0093] Amplification: Pipette 10 ng of DNA and prepare the amplification system according to Table 1. Use primer set C3, make up to 50 µL with water, and run PCR at 95°C for 3 min; (35 cycles of 95°C for 15 sec; 58°C for 15 sec; 72°C for 10 min); 72°C for 5 min; and hold at 4°C. After amplification, store at -20°C for 1 month.

[0094] Purification: Add 50 µL of DNA magnetic beads to the above reaction system, let it stand at room temperature for 10 minutes, place it on a magnetic stand for adsorption for 5 minutes, discard the supernatant, wash it twice with 80% ethanol, dry it at room temperature for 5 minutes, elute it with 47 µL of water, and take 1 µL of the product for quantification;

[0095] End Repair Plus "A": Take 45 µL of the above reaction system (Table 5), add 15 µL of End Repair Plus "A" reaction solution, and run PCR at 20°C for 10 min, 65°C for 10 min, and 4°C for hold.

[0096] Purification: Add 60µL DNA magnetic beads to the above reaction system, incubate at room temperature for 10 minutes, place on a magnetic stand for 5 minutes, discard the supernatant, wash twice with 80% ethanol, dry at room temperature for 5 minutes, elute with 23µL water, and take 1µL of the product for quantification;

[0097] Ligation Index: Take 21 µL of the above reaction system (Table 5), add 5 µL of ligation barcode BL01 to the above reaction system, add 14 µL of ligation reaction solution 1, and run PCR at 25°C for 30 min.

[0098] Purification: Add 2 µL of EDTA to the above reaction system and add 0.4× volume of DNA magnetic beads. After 10 min at room temperature, place on a magnetic stand for 5 min and discard the supernatant. Wash twice with 80% ethanol and dry at room temperature for 5 min. Elute with 32 µL of water and take 1 µL for quantification.

[0099] Adapter ligation: Add 5 µL of sequencing adapters and 70 µL of Ligation Reaction 2 (Table 5) to the above reaction system and run PCR at 25°C for 30 min.

[0100] Purification: Add 80µL of DNA magnetic beads to the above reaction system, incubate at room temperature for 10 minutes, place on a magnetic stand for 5 minutes, and discard the supernatant. Wash twice with 300µL of long fragment cleaning solution, then elute with 42µL of water. Take 1µL for quantification. If the total amount of product is greater than 1500ng, take 50ng for electrophoresis. The electrophoresis results show bands corresponding to 3000bp-12000bp ( Figure 10 ).

[0101] Sequencing and data analysis: The libraries were merged according to the BGI third-generation sequencing process. The amount of data off the machine was about 0.2G, which met the requirements. Analysis was carried out according to the human HLA third-generation sequencing analysis process V1.

[0102] Primer set C3 can be used to amplify HLA-II class genes. After index splitting, the target data volume is about 0.2G, which meets the expectations and analysis requirements ( Figure 11 ).

[0103] Compared with the control (PCR method to detect SNP), the typing results of primer set C3 were completely consistent with the results of the control for HLA-II DRB1 and DQB1, and the number of supported reads was more than 300 ( Figure 12 ).

[0104] The results of this example show that the C3 primer set can also amplify HLA-II class genes well, and the present invention is flexible to use, can adapt to the detection of HLA-II class genes, meet different needs, and is easy to operate.

[0105] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A set of primers for detecting HLA-I and HLA-II genes, characterized in that: The primers include primers shown in SEQ ID No.2-SEQ ID No.17, SEQ ID No.26, SEQ ID No.27, SEQ ID No.34, SEQ ID No.35, SEQ ID No.42, and SEQ ID No.43 in the sequence listing.

2. A kit for detecting HLA-I and HLA-II genes, characterized in that: The kit comprises the primers according to claim 1.

3. An HLA genotyping kit based on a third-generation sequencing technology platform, comprising the primers according to claim 1.

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