Molecular marker combination for detecting HLA-A*02: 01 and application thereof

Abstract

The invention discloses a molecular marker combination for detecting HLA-A * 02: 01 and application of the molecular marker combination. The molecular marker combination comprises a first molecular marker, a second molecular marker, a third molecular marker, a fourth molecular marker, a fifth molecular marker, a sixth molecular marker and a seventh molecular marker, and a primer group and a probe are designed for the molecular marker combination. The molecular marker combination for detecting the HLA-A * 02: 01, provided by the invention, can be effectively used for detecting the HLA-A * 02: 01, and is high in sensitivity and good in specificity. The reagent for detecting the molecular marker combination is designed aiming at the molecular marker combination, is good in anti-interference effect, high in sensitivity, good in specificity and short in detection time, and can be effectively used for detecting the HLA-A * 02: 01.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a reagent for detecting HLA-A*0201 and an application thereof. Background Art

[0002] HLA antigens are expressed by the human Major Histocompatibility Complex (MHC). In the immune system, they are primarily responsible for cell-to-cell recognition, inducing immune responses, and regulating immune responses. Analyzing HLA antigen expression not only helps understand disease pathogenesis but also has important implications for disease diagnosis, prevention, and prognosis.

[0003] The HLA gene is located on the short arm of chromosome 6, at position 6p21.31, and is approximately 3.6 Mb in length. HLA represents the most allelic polymorphism of any known human gene complex. Currently, over 40,623 HLA and related alleles are described using the HLA nomenclature. HLA-A*02:01 is one of the most common HLA-A alleles. Its sequence differs minimally from that of HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, and HLA-A*02:10, with high homology.

[0004] Currently, the HLA-A*02:01 allele is primarily identified through direct sequencing. This method can produce precise sequence information. However, sequencing methods are complex, costly, and require a long testing cycle, making them unsuitable for rapid disease diagnosis. Fluorescence PCR, on the other hand, utilizes primers complementary to the HLA allele sequence to perform specific PCR amplification of the DNA from the test sample. This method is low-cost, time-efficient, and highly specific, meeting clinical requirements for rapid testing and making it the most commonly used method for rapid single-gene testing. However, human HLA alleles exhibit extremely high homology and minimal sequence variation, making HLA-A*02:01 gene detection challenging. At least seven sites must be tested to distinguish HLA-A*02:01 from the more frequent HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, and HLA-A*02:53N alleles. The human genome consists of two sets of genes, and the conventional multiplex fluorescent PCR method's multiple site analysis is difficult to avoid crossover between the two sets of genes. Therefore, even if seven sites are tested, it is impossible to distinguish the nonspecificity caused by crossover between the two sets of genes.

[0005] Therefore, currently, no other rapid, highly specific methods for detecting HLA-A*02:01 are available on the market, aside from sequencing methods, which are complex, expensive, and require long detection cycles. This application proposes to develop a low-cost, easy-to-use, and minimally interfering fluorescence PCR method for detecting HLA-A*02:01, thereby addressing the challenges of detecting HLA-A*02:01. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a molecular marker combination for detecting HLA-A*02:01.

[0007] The present invention also provides a primer set and / or probe for detecting the above-mentioned HLA-A*02:01 molecular marker combination.

[0008] The present invention also provides the application of the above molecular marker combination, primer set and / or probe.

[0009] The present invention also provides a reagent for detecting HLA-A*02:01.

[0010] The present invention also provides a kit.

[0011] The present invention also provides the application of the above reagent or kit.

[0012] The present invention also provides a method for detecting HLA-A*02:01 for non-disease diagnosis purposes.

[0013] The present invention also provides a system for detecting HLA-A*02:01.

[0014] According to one aspect of the present invention, a molecular marker combination for detecting HLA-A*02:01 is provided, wherein the molecular marker combination includes a first molecular marker, a second molecular marker, a third molecular marker, a fourth molecular marker, a fifth molecular marker, a sixth molecular marker, and a seventh molecular marker;

[0015] The first molecular marker is rs2075684, and the polymorphism is A / T;

[0016] The second molecular marker is rs1059418, and the polymorphism is A / C;

[0017] The third molecular marker is rs199474457, and the polymorphism is C / T;

[0018] The fourth molecular marker is rs1136683, and the polymorphism is C / G;

[0019] The fifth molecular marker is rs41562119, and the polymorphism is C / G;

[0020] The sixth molecular marker is rs1136697, and the polymorphism is A / G / T;

[0021] The seventh molecular marker is rs9260156, and the polymorphism is T / G.

[0022] In some embodiments of the present invention, the first molecular marker, the second molecular marker, the third molecular marker, the fourth molecular marker, the fifth molecular marker, the sixth molecular marker and the seventh molecular marker are located on chromosome 6 of the GRCh38.p14 reference genome.

[0023] According to a second aspect of the present invention, a primer set and / or probe for detecting the above-mentioned molecular marker combination of HLA-A*02:01 is provided.

[0024] In some embodiments of the present invention, the primer set includes a first primer set and a second primer set;

[0025] The first primer set includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 16;

[0026] The second primer set includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO: 5 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO: 13.

[0027] In some embodiments of the present invention, the sequence of the probe is shown in SEQ ID NO:9.

[0028] In some embodiments of the present invention, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0029] In some embodiments of the present invention, the fluorescent reporter group includes but is not limited to any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC and TET.

[0030] In some embodiments of the present invention, the fluorescence quenching group includes but is not limited to any one of super Quenther1, BHQ, TAMRA, MGB and DABCYL.

[0031] According to a third aspect of the present invention, the use of the above-mentioned molecular marker combination, primer set and / or probe in any of the following is proposed:

[0032] (1) Detection of HLA genes;

[0033] (2) Preparation of products for detecting or assisting in the detection of HLA genes;

[0034] (3) Detect or differentiate HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, and / or HLA-A*02:53N genotypes;

[0035] (4) Preparation of products for detecting or differentiating HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11 and / or HLA-A*02:53N genotypes; such applications are not intended for the diagnosis or treatment of a disease.

[0036] According to a fourth aspect of the present invention, a reagent for detecting HLA-A*02:01 is provided, wherein the reagent comprises the above-mentioned primer set and / or probe.

[0037] In some embodiments of the present invention, the primer set includes a first primer set and a second primer set;

[0038] The first primer set includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 16;

[0039] The second primer set includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO: 5 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO: 13.

[0040] In some embodiments of the present invention, the sequence of the probe is shown in SEQ ID NO:9.

[0041] In some embodiments of the present invention, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0042] In some embodiments of the present invention, the fluorescent reporter group includes but is not limited to any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC and TET.

[0043] In some embodiments of the present invention, the fluorescence quenching group includes but is not limited to any one of super Quenther1, BHQ, TAMRA, MGB and DABCYL.

[0044] In some embodiments of the present invention, the reagent further comprises a PCR reaction solution; the PCR reaction solution comprises a stabilizer, a DNA polymerase, Mg 2+ and at least one of dNTPs.

[0045] In some embodiments of the present invention, the DNA polymerase is preferably a hot-start Taq DNA polymerase.

[0046] In some embodiments of the present invention, the PCR reaction solution is PCR mix.

[0047] In some embodiments of the present invention, the reagent further includes a primer probe set for detecting an internal reference gene. In the primer probe set for detecting an internal reference gene, the primer sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22; the probe sequence is shown in SEQ ID NO: 23.

[0048] According to a fifth aspect of the present invention, a kit is provided, which contains the above reagents or the above primer set and / or probe.

[0049] In a sixth aspect of the present invention, the application of the above-mentioned detection reagent and kit is proposed, wherein the application is the application in preparing a product for detecting or assisting in the detection of HLA-A*02:01.

[0050] In some embodiments of the present invention, the product is a kit or a chip.

[0051] In some embodiments of the present invention, the method of using the product is as follows:

[0052] 1) Extracting DNA from the sample to be tested;

[0053] 2) using the sample DNA to be tested as a template, performing a first round of PCR amplification reaction using a first primer set to obtain a first amplification product;

[0054] 3) using the amplified product as a template, performing a second round of PCR amplification reaction using a second primer set and a probe to obtain a second amplified product;

[0055] 4) Analyze the second amplification product.

[0056] In some embodiments of the present invention, the reaction system used in the first round of PCR amplification reaction is as follows:

[0057]

[0058]

[0059] Add ddH2O to make up to 20 μL.

[0060] In some embodiments of the present invention, the reaction system used in the second round of PCR amplification reaction is as follows:

[0061]

[0062] Add ddH2O to make up to 20 μL.

[0063] In some embodiments of the present invention, the reaction system used in the second round of PCR amplification reaction also includes a primer probe set for detecting an internal reference gene. In the primer probe set for detecting an internal reference gene, the primer sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22; the probe sequence is shown in SEQ ID NO: 23.

[0064] In some embodiments of the present invention, the primer-probe set for detecting the internal reference gene in the reaction system used in the second round of PCR has a primer concentration of 0.05-0.5 μM and a probe concentration of 0.05-0.5 μM.

[0065] In some embodiments of the present invention, the reaction procedure of the first round of PCR amplification reaction is: 94-96°C for 3-7 min; then enters the cycling stage: 93-96°C for 8-12 s, 58-62°C for 20-30 s, and 68-74°C for 30-40 s for 14-20 cycles.

[0066] In some embodiments of the present invention, the reaction procedure of the second round of PCR amplification reaction is: 94-96° C. for 3-7 min; then enter the cycling stage: 93-96° C. for 8-12 s, 58-64° C. for 20-30 s, for 35-45 cycles, and collect fluorescence data.

[0067] In some embodiments of the present invention, the analyzing of the second amplification product includes: performing statistics on the Ct value of the sample and interpreting the result; the result interpretation is:

[0068] Invalid: If the Ct of the internal reference is ≥36, the test result is invalid;

[0069] Positive: If the Ct of the internal reference is less than 36, and the ΔCT between the Ct of the sample and the internal reference gene is less than 4, the sample is positive and carries the HLA-A*02:01 allele;

[0070] Negative: If the Ct of the internal reference is less than 36, and the Ct of the sample and the ΔCT of the internal reference gene are ≥ 4, the sample is negative and does not carry the HLA-A*02:01 allele.

[0071] In a seventh aspect of the present invention, a method for detecting HLA-A*02:01 for non-disease detection purposes is provided, the method comprising the following steps: using the above-mentioned reagent or kit to detect the DNA of a sample to be tested.

[0072] In some embodiments of the present invention, the detection specifically comprises the following steps:

[0073] 1) Using the sample DNA to be tested as a template, a first round of PCR amplification reaction is performed using a first primer set to obtain a first amplification product;

[0074] 2) Using the amplified product as a template, a second round of PCR amplification reaction is performed using a second primer set and a probe to obtain a second amplified product.

[0075] In some embodiments of the present invention, the reaction system used in the first round of PCR amplification reaction is as follows:

[0076]

[0077] Add ddH2O to make up to 20 μL.

[0078] In some embodiments of the present invention, the reaction system used in the second round of PCR amplification reaction is as follows:

[0079]

[0080] Add ddH2O to make up to 20 μL.

[0081] In some embodiments of the present invention, the reaction system used in the second round of PCR amplification reaction also includes a primer probe set for detecting an internal reference gene. In the primer probe set for detecting an internal reference gene, the primer sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22; the probe sequence is shown in SEQ ID NO: 23.

[0082] In some embodiments of the present invention, the primer-probe set for detecting the internal reference gene in the reaction system used in the second round of PCR has a primer concentration of 0.05-0.5 μM and a probe concentration of 0.05-0.5 μM.

[0083] In some embodiments of the present invention, the reaction procedure of the first round of PCR amplification reaction is: 94-96°C for 3-7 min; then enter the cycling stage: 93-96°C for 8-12 s, 58-62°C for 20-30 s, 68-74°C for 30-40 s for 14-20 cycles, and collect fluorescence data.

[0084] In some embodiments of the present invention, the reaction procedure of the second round of PCR amplification reaction is: 94-96° C. for 3-7 min; then enter the cycling stage: 93-96° C. for 8-12 s, 58-64° C. for 20-30 s, for 35-45 cycles, and collect fluorescence data.

[0085] In some embodiments of the present invention, after the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is further included; the result interpretation is:

[0086] Invalid: If the Ct of the internal reference is ≥36, the test result is invalid;

[0087] Positive: If the Ct of the internal reference is less than 36, and the ΔCT between the Ct of the sample and the internal reference gene is less than 4, the sample is positive and carries the HLA-A*02:01 allele;

[0088] Negative: If the Ct of the internal reference is less than 36, and the Ct of the sample and the ΔCT of the internal reference gene are ≥ 4, the sample is negative and does not carry the HLA-A*02:01 allele.

[0089] According to an eighth aspect of the present invention, a system for detecting HLA-A*02:01 is provided, the system comprising:

[0090] An amplification module is used to perform PCR amplification on the genomic DNA of the sample to be tested using the above reagents or kit to obtain an amplified product;

[0091] The result evaluation module is used to detect the amplification product and evaluate whether the sample to be tested carries the HLA-A*02:01 allele.

[0092] According to some embodiments of the present invention, the system further comprises a DNA template extraction module for extracting genomic DNA from the sample to be tested.

[0093] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the molecular marker combination for detecting HLA-A*02:01 provided by the present invention can be effectively used for detecting HLA-A*02:01 with high sensitivity and good specificity. A reagent designed for detecting the molecular marker combination exhibits excellent anti-interference properties, high sensitivity, good specificity, and a short detection time, and can be effectively used for detecting HLA-A*02:01.

[0094] The method for detecting HLA-A*02:01, based on the aforementioned HLA-A*02:01 reagent, utilizes two rounds of PCR, nested nested PCR, to simultaneously detect seven loci within a single human HLA-A*02:01 gene sequence. This avoids the nonspecificity inherent in conventional PCR methods, which cannot distinguish between polyploid gene sequences and result in crossovers. It also eliminates the nonspecificity associated with the high-frequency HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, and HLA-A*02:53N allele combinations, as well as other common HLA-A alleles. Therefore, this method effectively addresses the challenges of HLA-A*02:01 gene detection, which are often difficult to detect using conventional multiplex PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0096] Figure 1 This is a graph showing the results of primer screening for site 1+site 2 in an embodiment of the present invention;

[0097] Figure 2 This is a graph showing the results of primer screening for site 3+site 4 in an embodiment of the present invention;

[0098] Figure 3 This is a diagram showing the results of the probe screening for site 5 in an embodiment of the present invention;

[0099] Figure 4 This is a primer screening result diagram for site 6 in an embodiment of the present invention;

[0100] Figure 5 This is a primer screening result diagram for site 7 in an embodiment of the present invention;

[0101] Figure 6 Graph showing the amplification effect test results of Procedure 1 (58° C.) and Procedure 2 (62° C.) in the embodiments of the present invention;

[0102] Figure 7 Graph showing the amplification effect test results of Procedure 1 (58° C.) and Procedure 2 (64° C.) in the embodiments of the present invention;

[0103] Figure 8 Graph showing the amplification effect test results of Procedure 1 (60° C.) and Procedure 2 (62° C.) in an embodiment of the present invention;

[0104] Figure 9 Graph showing the amplification effect test results of Procedure 1 (60° C.) and Procedure 2 (64° C.) in the embodiments of the present invention;

[0105] Figure 10 Graph showing the amplification effect detection results of Procedure 1 (62° C.) and Procedure 2 (62° C.) in the examples of the present invention. DETAILED DESCRIPTION

[0106] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0107] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0108] Example 1 Molecular marker combination for detecting HLA-A*02:01 and design of primers and probes for detecting the molecular marker combination

[0109] (1) Selection of molecular marker combinations

[0110] Through bioinformatics analysis, the present invention screened and compared seven specific loci as shown in Table 1 below (site 1: rs2075684, site 2: rs1059418, site 3: rs199474457, site 4: rs1136683, site 5: rs41562119, site 6: rs1136697, and site 7: rs9260156). All of these loci are located on chromosome 6 of the GRCh38.p14 reference genome. These loci can distinguish common HLA genes as well as HLA genotypes with extremely high sequence similarity, such as HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, and HLA-A*02:53N.

[0111] From the perspective of a single allele, differentiation can be achieved through these 7 specific sites, but the human genome consists of two sets of genes, and the multiple site analysis of conventional multiplex fluorescence PCR methods is difficult to avoid the nonspecificity caused by the crossover of the two sets of genes.

[0112] Analysis of differential allele sites for HLA-A*02 is shown in Table 1 below. HLA-A*02:06 is specific for sites 1 and 2 compared to HLA-A*02:01, and HLA-A*02:07 is specific for site 6 compared to HLA-A*02:01. However, using conventional multiplex fluorescent PCR methods, primer-probe systems developed for sites 1, 2, and 6, or all seven specific sites, can still result in false positives for HLA-A*02:06 / HLA-A*02:07 genotypes because the two sets of genes do not share a common site.

[0113] Table 1 Analysis of differential sites of HLA-A*02 alleles

[0114] Allele Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 A*02:01 T A C C C A T A*02:07 T A C C C G T A*02:06 A C C C C A T A*02:03 T A C C C A G A*02:10 A C C C C T T A*02:05 A C C C C A G A*02:53N T A C C G A T A*02:11 T A T G C A T

[0115] (2) Design of primers and probes

[0116] Therefore, to ensure simultaneous detection of seven loci within a single gene sequence and avoid cross-talk between the two gene sets, this protocol employed a specialized primer-probe design method, utilizing biological software to design specific primer-probes for each of the seven loci. Comprehensive analysis of the multiple pairs of designed primer-probes subsequently resulted in the selection of the primer-probe sets shown in Table 2 below. These sequences were synthesized by General Biotech (Anhui) Co., Ltd.

[0117] Table 2 Primer sequence information

[0118]

[0119]

[0120] (3) Optimization of primer probe sets

[0121] Using the different primer probes designed for each site in step (1), a primer probe screening experiment is performed. By comparing the amplification effects of different primer probes on positive plasmids and the exclusion effects of non-specific plasmids, the primers and probes with the best detection effects are screened.

[0122] The design and preparation steps of the detection template were as follows: a positive plasmid was constructed based on the original HLA-A*0201 gene sequence (SEQ ID NO:24). Nonspecific plasmids were designed based on the original HLA-A*0201 gene sequence for each site (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29) to construct nonspecific plasmids (plasmid 1 for specific sites 1-2 (HLA-A*02:06), plasmid 2 for specific sites 3-4 (HLA-A*02:11), plasmid 3 for specific site 5 (HLA-A*02:53N), plasmid 4 for specific site 6 (HLA-A*02:07), and plasmid 5 for specific site 7 (HLA-A*02:03). The plasmids were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0123] Table 3 Gene sequences inserted into positive plasmids and nonspecific plasmids

[0124]

[0125]

[0126]

[0127]

[0128] After diluting each plasmid to 0.5 ng / mL, each specific site was tested using the reaction system in Table 4 and the reaction procedure in Table 5. The quality of the primer and probe design was determined based on the test results.

[0129] Table 4 Primer probe screening reaction system

[0130]

[0131]

[0132] Table 5 Primer probe screening PCR reaction program

[0133]

[0134] The screening of primer probes was carried out in the order of site 1+site 2, site 3+site 4, site 6, site 7, and site 5.

[0135] First round of screening: SEQ ID NO: 1-SEQ ID NO: 4 of site 1 + site 2 were used as forward primers, SEQ ID NO: 14 of site 7 was used as a reverse primer, and SEQ ID NO: 8 of site 5 was used as a probe, and SEQ ID NO: 1 of site 1 + site 2 was obtained by screening. Second round of screening: SEQ ID NO: 5-SEQ ID NO: 7 of site 3 + site 4 were used as forward primers, SEQ ID NO: 10 of site 6 was used as a reverse primer, and SEQ ID NO: 8 of site 5 was used as a probe, and SEQ ID NO: 5 of site 3 + site 4 was obtained by screening. Third round of screening: SEQ ID NO: 5 of site 3 + site 4 were used as forward primers, SEQ ID NO: 10-SEQ ID NO: 13 of site 6 was used as a reverse primer, and SEQ ID NO: 8 of site 5 was used as a probe, and SEQ ID NO: 13 of site 6 was obtained by screening. Fourth round of screening: SEQ ID NO: 1 at site 1 + site 2 was used as a forward primer, SEQ ID NO: 14-SEQ ID NO: 20 at site 7 was used as a reverse primer, and SEQ ID NO: 8 at site 5 was used as a probe, resulting in SEQ ID NO: 16 at site 7. Fifth round of screening: SEQ ID NO: 5 at site 3 + site 4 was used as a forward primer, SEQ ID NO: 13 at site 6 was used as a reverse primer, and SEQ ID NO: 8-SEQ ID NO: 9 at site 5 was used as a probe, resulting in SEQ ID NO: 9 at site 5.

[0136] Test results such as Figure 1-5 As shown in the figure, it can be seen that the best result is obtained by screening SEQ ID NO: 1 with site 1 + site 2; the best result is obtained by screening SEQ ID NO: 5 with site 3 + site 4; the best result is obtained by screening SEQ ID NO: 9 with site 5; the best result is obtained by screening SEQ ID NO: 13 with site 6; and the best result is obtained by screening SEQ ID NO: 16 with site 7. SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 16 were subsequently selected for further system optimization.

[0137] (4) Reaction program optimization

[0138] Based on the TM value prediction of bioinformatics analysis and the experimental results of step (2), the reaction program of the primer probe obtained by screening was optimized.

[0139] The PCR amplification process is divided into two steps. The first round of PCR reaction program is shown in Table 6-7 below. Three temperatures, 58°C, 60°C, and 62°C, are selected for comparative analysis. After the first round of PCR, a second round of PCR is performed at two temperatures, 62°C and 64°C.

[0140] Table 6 First round PCR reaction program

[0141]

[0142] Table 7 Second round PCR reaction program

[0143]

[0144] The reaction systems used for the two rounds of PCR are shown in Tables 8-9 below. The reaction system components were prepared according to the required amounts, mixed thoroughly, and capped for PCR amplification and detection. The templates used in the first round of PCR were an HLA-A*0201-positive plasmid and five plasmids containing specific loci. The template used in the second round of PCR was the product from the first round of PCR.

[0145] Table 8 Component A of the first round PCR reaction solution

[0146]

[0147]

[0148] Table 9 Components of the second round PCR reaction solution B

[0149]

[0150] The results are as follows Figure 6-10 As shown in the figure, when the first and second rounds of PCR were performed at 62°C, the positive plasmid amplification effect was better. Furthermore, the difference in CT values ​​between the positive and nonspecific plasmids was the largest, nonspecific interference was minimized, and discrimination was the best. This protocol was selected for further experiments.

[0151] Example 2 A kit for detecting HLA-A*02:01

[0152] This example provides a kit for detecting HLA-A*02:01, which contains the primer sequences screened in Example 1 (specific sequences are shown in Table 10), each primer at a concentration of 10 μM, PCR mix, negative control, and positive control.

[0153] Among them, the negative control was ultrapure water;

[0154] The positive control was the positive plasmid constructed using the original HLA-A*0201 gene sequence (SEQ ID: 24) in Example 1.

[0155] Table 10

[0156]

[0157]

[0158] The method for detecting HLA-A*02:01 using the kit is as follows:

[0159] (1) According to the instructions of the nucleic acid extraction or purification reagent GMB-MS-P200 of Changzhou Jinmai Biotechnology Co., Ltd., nucleic acid was extracted from the test samples, negative controls and positive controls.

[0160] (2) Prepare the PCR reaction solution A component with reference to Table 8. 1 μL of the processed sample nucleic acid, negative control, and positive control were aspirated as templates, and added to the first-round PCR reaction solution A. The mixture was fully mixed to form a PCR mixture. The tube was capped and the first-round PCR amplification was performed on a fluorescent PCR instrument. The system used for the first-round PCR amplification was shown in Table 8, and the reaction procedure was shown in Table 6 (the temperature was 62°C). The second-round PCR reaction system used the first-round PCR reaction product as the detection template. The reaction system used was shown in Table 9, and the reaction procedure was shown in Table 7 (the fluorescence collection temperature was 62°C).

[0161] Result analysis:

[0162] After the reaction is complete, the results are automatically saved and the amplification curves of the detected targets are analyzed separately. Based on the analyzed image, adjust the Baseline Start, End, and Threshold values ​​(users can adjust the Start value to 3-15 and the End value to 5-20 based on actual conditions. Adjust the negative control amplification curve to make it flat or below the threshold line). Click Analyze to ensure that all parameters meet the requirements of "Quality Control" below. Then, record the qualitative results in the Plate window.

[0163] Quality Control:

[0164] Negative control: no Ct value in FAM and VIC channels;

[0165] Positive control: FAM and VIC channels both Ct≤36, ΔCT<4;

[0166] The above requirements must be met simultaneously in the same experiment; otherwise, the experiment will be invalid and must be repeated.

[0167] Positive judgment value:

[0168] The Ct reference value of the target gene detected by this kit was determined to be 36 through reference value research.

[0169] Interpretation of test results

[0170] Evaluation of test results from clinical specimens should be performed after positive and negative controls have been tested and determined to be valid and acceptable. If the controls are invalid, interpretation of patient results is impossible. Table 11 describes the interpretation of results using the above controls. End users should review the fluorescence curves before final interpretation.

[0171] Table 11 HLA-A*02:01 test result determination method

[0172]

[0173] Example 3 Simulated sample detection

[0174] The kit prepared in Example 2 and the detection method provided were used to perform fluorescence PCR detection on the single type of plasmid synthesized in Example 1 and the plasmid containing the five specific sites, which were mixed in a 1:1 ratio.

[0175] Table 12 Single type plasmid detection results

[0176]

[0177] Table 13 Results of plasmid 1:1 mixing

[0178]

[0179]

[0180] The plasmid detection results are shown in Tables 12 and 13. As can be seen from the tables, the detection ΔCT of single-type and mixed plasmid simulation samples with HLA-A*02:01 positive plasmids is greater than 8, and the mixed plasmids of single-type and simulation samples can be effectively distinguished from the positive plasmids.

[0181] Example 4: Real Sample Detection and Judgment Criteria Analysis

[0182] The kit prepared in Example 2 and the provided detection method were used to perform fluorescence PCR detection on 10 clinical samples with sequencing results (blood samples from the Third Xiangya Hospital of Central South University).

[0183] Table 14 HLA-A*02:01 real sample detection results

[0184]

[0185] The results are shown in Table 14. As can be seen from the table, HLA-A*02:06 / HLA-A*02:07 were not detected, which can be clearly distinguished from HLA-A*02:01 positive, which is consistent with the actual results. The results show that based on the plasmid detection results of Example 3 and the actual sample detection results of this example, ΔCT < 4 can be preliminarily used as the threshold for positive-negative determination.

[0186] Example 5 Accuracy Detection

[0187] Using the kit prepared in Example 2 and the provided detection method, fluorescent PCR detection was performed on 20 clinical samples with sequencing results that were completely different from the clinical samples used in Example 4 (blood samples from the Third Xiangya Hospital of Central South University).

[0188] Table 15 HLA-A*02:01 real sample accuracy test results

[0189]

[0190]

[0191] The clinical sample test results are shown in Table 15. The test results of the 20 HLA-A type samples all met the requirements, and the consistency rate with the sequencing results was 100%.

[0192] Example 6: Repeatability and sensitivity testing

[0193] Using the kit prepared in Example 2 and the provided detection method, 10 PCR repeats were performed on two 10 ng / μL HLA-A*02:01 clinical samples (blood samples from the Third Xiangya Hospital of Central South University) with sequencing results.

[0194] Table 16 Sensitivity test results

[0195]

[0196]

[0197] The results, shown in Table 16, showed that the composition of the present invention could accurately detect samples as low as 10 ng / μL, with a positive detection rate of 100%, demonstrating that the sensitivity can reach below 10 ng / μL. Furthermore, the detection CV was less than 2% at a sample nucleic acid concentration of 10 ng / μL, indicating stable test results.

[0198] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A molecular marker combination for detecting HLA-A*02:01, characterized in that: The molecular marker combination includes a first molecular marker, a second molecular marker, a third molecular marker, a fourth molecular marker, a fifth molecular marker, a sixth molecular marker and a seventh molecular marker; The first molecular marker is rs2075684, and the polymorphism is A / T; The second molecular marker is rs1059418, and the polymorphism is A / C; The third molecular marker is rs199474457, and the polymorphism is C / T; The fourth molecular marker is rs1136683, and the polymorphism is C / G; The fifth molecular marker is rs41562119, and the polymorphism is C / G; The sixth molecular marker is rs1136697, and the polymorphism is A / G / T; The seventh molecular marker is rs9260156, and the polymorphism is T / G.

2. A primer set and / or probe for detecting the HLA-A*02:01 molecular marker combination according to claim 1.

3. Use of the molecular marker combination according to claim 1 or the primer set and / or probe according to claim 2 in any of the following: (1) Detection of HLA genes; (2) Preparation of products for detecting or assisting in the detection of HLA genes; (3) Detect or differentiate HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, and / or HLA-A*02:53N genotypes; (4) Preparation of products for detecting or distinguishing HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11 and / or HLA-A*02:53N genotypes.

4. A reagent for detecting HLA-A*02:01, characterized in that The reagents include the primer set and / or probe according to claim 2; Preferably, the primer set includes a first primer set and a second primer set; The first primer set includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 16; The second primer set includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13; Preferably, the sequence of the probe is shown in SEQ ID NO: 9; More preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; More preferably, the fluorescent reporter group includes but is not limited to any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC and TET; More preferably, the fluorescence quenching group includes but is not limited to any one of super Quenther 1, BHQ, TAMRA, MGB and DABCYL.

5. The reagent according to claim 1, characterized in that The reagent also includes a PCR reaction solution; the PCR reaction solution includes a stabilizer, DNA polymerase, Mg 2+ and at least one of dNTPs; And / or, the reagent further comprises a primer probe set for detecting an internal reference gene; In the primer-probe set for detecting the internal reference gene, the primer sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22, and the probe sequence is shown in SEQ ID NO:

23.

6. A kit, characterized in that The kit comprises the primer set and / or probe according to claim 2 or the reagent according to any one of claims 4-5.

7. Use of the reagent according to any one of claims 4 to 5 or the kit according to claim 6 in the preparation of a product for detecting or assisting in the detection of HLA-A*02:

01.

8. The use according to claim 7, characterized in that The method of using the product is as follows: 1) Extracting DNA from the sample to be tested; 2) using the sample DNA to be tested as a template, performing a first round of PCR amplification reaction using a first primer set to obtain a first amplification product; 3) using the amplified product as a template, performing a second round of PCR amplification reaction using a second primer set and a probe to obtain a second amplified product; 4) analyzing the second amplification product; Preferably, the reaction system used in the first round of PCR amplification reaction is as follows: Add ddH2O to 20 μL; Preferably, the reaction system used in the second round of PCR amplification reaction is as follows: Add ddH2O to 20 μL; Preferably, the reaction program of the first round of PCR amplification reaction is: 94-96°C for 3-7 minutes; then enter the cycling stage: 93-96°C for 8-12 seconds, 58-62°C for 20-30 seconds, and 68-74°C for 30-40 seconds for 14-20 cycles; Preferably, the reaction program of the second round of PCR amplification reaction is: 94-96°C for 3-7 min; then enter the cycling stage: 93-96°C for 8-12 s, 58-64°C for 20-30 s, 35-45 cycles, and collect fluorescence data. Preferably, the analysis of the second amplification product includes: performing statistics on the Ct value of the sample and interpreting the result; the result interpretation is: Invalid: If the Ct of the internal reference is ≥36, the test result is invalid; Positive: If the Ct of the internal reference is less than 36, and the ΔCT between the Ct of the sample and the internal reference gene is less than 4, the sample is positive and carries the HLA-A*02:01 allele; Negative: If the Ct of the internal reference is less than 36, and the Ct of the sample and the ΔCT of the internal reference gene are ≥ 4, the sample is negative and does not carry the HLA-A*02:01 allele.

9. A method for detecting HLA-A*02:01 for non-disease detection purposes, characterized in that: The method comprises the following steps: using the reagent according to any one of claims 4 to 5 or the kit according to claim 6 to detect the DNA of the sample to be tested.

10. A system for detecting HLA-A*02:01, characterized in that The system comprises: an amplification module, configured to perform PCR amplification on the genomic DNA of the sample to be tested using the reagent according to any one of claims 4 to 5 or the kit according to claim 6 to obtain an amplified product; The result evaluation module is used to detect the amplification product and evaluate whether the sample to be tested carries the HLA-A*02:01 allele.

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