Primers, kits, and methods for high-resolution genotyping of MICA and MICB genes.

Abstract

This invention belongs to the field of gene detection technology and discloses primers, kits, and methods for high-resolution genotyping of MICA and MICB genes. The primers include primer pairs SEQ ID NO:1 and SEQ ID NO:2 for the MICA gene and primer pairs SEQ ID NO:3 and SEQ ID NO:4 for the MICB gene. Using these primers, only one amplification is needed to simultaneously and effectively amplify the specific sequences of the MICA and MICB genes, thereby accurately analyzing the high-resolution genotyping of the MICA and MICB genes and effectively reducing the false positive rate and retesting rate.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to primers, kits, and methods for high-resolution genotyping of MICA and MICB genes. Background Technology

[0002] The MIC gene (MHC class I chain-related gene) is located on human chromosome 6 and is a gene family within the human leukocyte antigen (HLA) gene complex, belonging to the non-classical HLA-I class. The gene family contains seven members (MICA-MICG), of which only MICA and MICB are functional genes, located at 46kb and 140kb positions on the centromere, respectively. MICA and MICB genes exhibit extensive polymorphism, being the most polymorphic among all non-classical HLA genes. They are not expressed on lymphocytes and therefore cannot undergo cross-matching.

[0003] The MICA gene is 11722 bp (NM_000247) long, encoding a 1382 bp transcript; it encodes a membrane-bound protein of 383-389 amino acids with a molecular weight of 62 kD. The entire gene consists of 6 exons: exon 1 encodes the L leader peptide, exons 2-4 encode the extracellular α1, α2, and α3 domains, respectively, exon 5 encodes the transmembrane region, and exon 6 encodes the cytoplasmic region. The MICB gene is 12930 bp long, encoding a 2376 bp transcript; it encodes 383 amino acids. The coding regions of these two genes share 80%, 91%, 90%, 98%, 73%, and 82% homology, respectively. The leader peptide and α1 domain of both the MICA and MICB genes are separated by a large intron, 6840 bp and 7352 bp in length, respectively. According to the latest data (v 3.62) from the IPD-IMGT / HLA database, 589 MICA alleles and 312 MICB alleles have been identified.

[0004] Unlike classic MHC class I molecules, MICA and MICB do not bind to β2 microglobulin or have antigen peptide presentation functions. Instead, they are expressed under cellular stress, acting as ligands to specifically activate the key receptor NKG2D (KLRK1, also known as CD314) of the innate immune system, thereby initiating immune surveillance mechanisms. Under normal physiological conditions, MICA and MICB are almost not expressed on the surface of healthy cells; however, their expression is significantly upregulated when cells encounter pathological stimuli such as DNA damage, viral infection, heat shock, or oxidative stress. As ligands of NKG2D, MICA and MICB are mainly expressed on the surface of stressed cells. By binding to NKG2D on the surface of NK cells, CD8+ T cells, γδ T cells, and activated macrophages, they activate innate and adaptive immune responses, prompting effector cells to exert cytotoxicity and secrete cytokines (such as IFN-γ). This mechanism is particularly crucial in tumor immune surveillance; MICA and MICB-positive tumor cells can be recognized and eliminated by the immune system, forming the body's first line of defense against tumors. MICA and MICB are expressed at low levels in normal cells, but their expression levels are significantly increased in epithelial tumors. This expression can be induced by bacterial and viral infections, suggesting that MICA and MICB play important roles in tumor surveillance, viral infection, and autoimmune diseases.

[0005] MICA and MICB genes are among the most extensively studied non-classical HLA antigen targets. MICA and MICB genotyping is primarily used for matching in organ and bone marrow transplantation and for screening for susceptibility genes in autoimmune diseases. Multiple studies have shown that MICA and MICB genotype mismatch between donors and recipients is independently associated with an increased risk of allogeneic transplantation failure, with the correlation varying across different organ transplants. For example, in kidney transplantation, MICA and MICB are defined as true transplant antigens, and it is recommended that MICA and MICB identification be included in pre-transplant testing. Some studies have found that MICA and MICB incompatibility is associated with an increased incidence of GVHD, and pre-transplant MICA and MICB genotyping is helpful in assessing the risk of GVHD. Understanding the polymorphisms of the MICA and MICB genes and performing MICA and MICB gene matching between donors and patients before organ transplantation is of great significance.

[0006] Furthermore, high expression of MICB is associated with a favorable prognosis in colorectal cancer, while its gene mutations may be associated with rheumatoid arthritis, cytomegalovirus and herpes simplex virus I seropositivity, and the risk of schizophrenia. The MICA gene is associated to varying degrees with genetic susceptibility and severity of various autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, psoriasis, and inflammatory bowel disease. Global genomic correlation analysis studies have shown that the MICA gene is a significantly significant risk allele in many patients with autoimmune diseases.

[0007] Common MICA genotyping methods include polymerase chain reaction (PCR-SSP) sequence-specific primer analysis; polymerase chain reaction (PCR-SSO) or SSOP oligonucleotide probe analysis; and polymerase chain reaction (PCR-SBT) direct nucleic acid sequencing. PCR-SSP interprets results based on the presence or absence of PCR amplification products. This method is highly sensitive and simple to operate, and is often used for preliminary genotyping. PCR-SSO can only genotype known alleles, is cumbersome and time-consuming, and is prone to false positives; it also cannot detect new alleles, requiring continuous kit updates. PCR-SBT, on the other hand, can directly read allele DNA sequences, offering high accuracy and the ability to discover new alleles. It is currently the internationally recognized gold standard for HLA genotyping. However, this method also suffers from high cost, long processing time, and low throughput, making it unsuitable for automated operation.

[0008] With the continuous development of next-generation sequencing (NGS), high-resolution HLA genotyping technology based on NGS has emerged and is now maturely applied to the genotyping of HLA-I / II class genes HLA-A / B / C / DRB1 / DQB1 / DPB1. It can simultaneously perform multi-sample, multi-site detection, offering high resolution, automated operation, simplicity, and stability, demonstrating significant technological advantages. However, for non-classical HLA-I class genes MICA and MICB, current research is primarily focused on scientific research, and a unified clinical consensus or guideline for detection has not yet been established. Currently, there are no commercially available kits on the market that simultaneously detect MICA and MICB genotyping based on NGS technology. The main technical challenges are: the coding regions of MICA and MICB genes are highly homologous (73%–98%), and there are a large number of single nucleotide polymorphisms (SNPs) and insertions / deletions (Indels). Short-read NGS sequencing is prone to mapping errors during sequence alignment, leading to difficulties in allele interpretation; paralogous sequence interference, with some sequences being similar to other genes in the HLA region, affecting specific capture or amplification; and the intron regions are long and polymorphic, making it difficult to accurately distinguish haplotypes if an exon-targeted capture scheme is used. Summary of the Invention

[0009] The purpose of this invention is to provide primers and kits for rapid detection of high-resolution genotyping of MICA and MICB genes, effectively improving the accuracy and resolution of high-resolution genotyping of MICA and MICB genes.

[0010] The following technical solutions are used to achieve the above objectives.

[0011] In a first aspect, the present invention provides primers for detecting high-resolution genotyping of MICA and MICB genes, the primers comprising:

[0012] Primer pairs targeting the MICA gene: SEQ ID NO:1 and SEQ ID NO:2;

[0013] Primer pairs SEQ ID NO:3 and SEQ ID NO:4 targeting the MICB gene.

[0014] Secondly, the present invention provides the application of the primers described above for detecting high-resolution genotyping of MICA and MICB genes in the detection of high-resolution genotyping of MICA and MICB genes.

[0015] Thirdly, the present invention provides a kit for high-resolution genotyping of MICA and MICB genes, the kit comprising the primers described above.

[0016] In some embodiments, the kit further includes PCR amplification reagents; the PCR amplification reagents include DNA polymerase and PCR buffer;

[0017] Preferably, the PCR amplification reagent includes Tks Gflex DNA Polymerase.

[0018] And / or, the PCR buffer contains Mg 2+ 2×Gflex PCR Buffer with dNTPs.

[0019] Fourthly, the present invention provides the application of the kit for high-resolution genotyping of MICA and MICB genes as described above in the detection of high-resolution genotyping of MICA and MICB genes.

[0020] Fifthly, the present invention provides a method for high-resolution genotyping of MICA and MICB genes, comprising the following steps:

[0021] Step 1. Mix the primer pair targeting the MICA gene and the primer pair targeting the MICB gene to prepare a mixed primer; the primer pair sequences targeting the MICA gene are shown in SEQ ID NO:1 and SEQ ID NO:2, and the primer pair sequences targeting the MICB gene are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0022] Step 2. Using the DNA sample to be tested as a template and the mixed primers from Step 1 as primers, perform PCR amplification to obtain the amplification product, and purify the obtained amplification product;

[0023] Step 3. Perform enzyme digestion, end repair, A base addition, and adapter ligation on the amplification products to construct a sequencing library;

[0024] Step 4. Sequencing the sequencing library.

[0025] In some embodiments, the working concentration of the primers is 0.5 μM to 5 μM.

[0026] In some embodiments, the working concentration of the primer is 0.5 μM to 1 μM, preferably 0.7 μM to 0.9 μM.

[0027] In some embodiments, in step 1, the primers in the mixed primers are mixed in equal proportions;

[0028] And / or, in step 3, the sequencing is performed using the Illumina sequencing platform;

[0029] And / or, in step 4, the library concentration is ≥1 ng / μL.

[0030] In some embodiments, the PCR amplification reaction procedure in step 2 is as follows:

[0031] Pre-denaturation: Treat at 93℃~95℃ for 1min~2min, 1 cycle;

[0032] Amplification cycle: 95℃~100℃ for 8 sec~12 sec, 65℃~70℃ for 4 min~6 min, for a total of 25 cycles;

[0033] And / or, the reaction system of the amplification reaction is as follows: a 25 μL reaction system containing 90 ng to 110 ng of DNA template, 0.5 to 1.5 μL of Tks Gflex DNA Polymerase, 10 to 15 μL of 2×Gflex PCR Buffer, 3 to 8 μL of mixed primers, and the remainder being water.

[0034] This invention provides a kit for the combined detection of MICA and MICB genotyping. By designing specific primers suitable for detecting MICA and MICB genotyping, the kit allows for the simultaneous and effective amplification of the specific sequences of the MICA and MICB genes in a single amplification step. This enables accurate analysis of the high-resolution genotyping of the MICA and MICB genes, effectively reducing the false positive rate and retest rate of the detection results.

[0035] This invention reveals that primer concentration and selection of appropriate amplification enzymes have a significant impact on obtaining sufficient amplification products while avoiding the formation of primer dimers. Based on the specific primers mentioned above, only by obtaining suitable PCR reaction conditions can specific amplification be achieved, thereby enabling accurate high-resolution genotyping detection.

[0036] The detection method of this invention uses specific primers, primer concentrations, and amplification enzymes to successfully amplify all specific base sequences in the MICA & MICB coding gene regions, construct libraries, and combine with high-throughput sequencing to effectively identify MICA and MICB gene sequences, thereby accurately analyzing high-resolution genotyping of MICA and MICB genes. The detection process is rapid, simple, high-throughput, and low-cost, suitable for large-scale matching and gene screening, and applicable to matching for organ and bone marrow transplantation and screening for susceptibility genes in autoimmune diseases. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the MICA and MICB genes.

[0038] Figure 2 This is a flowchart of a method for high-resolution genotyping of the MICA & MICB genes.

[0039] Figure 3 This is a flowchart of the bioinformatics analysis in Example 1.

[0040] Figure 4 This is an agarose gel electrophoresis of the primer set amplified in Example 2.

[0041] Figure 5 A is the result of testing a primer mixture with a concentration of 0.083 μM; Figure 5 B is the result of testing with a primer mixture at a concentration of 0.83 μM; Figure 5 C is the result of testing with a primer mixture at a concentration of 8.3 μM. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0045] It should be noted that in this article, MICA&MICB refers to the combined analysis of the MICA and MICB genes.

[0046] High-resolution genotyping: Using high-throughput sequencing technology, the allelic differences between MICA and MICB genes at the nucleotide level are accurately distinguished.

[0047] Example 1: Method and Flowchart for High-Resolution Genotyping of MICA & MICB Genes

[0048] Please refer to Figure 1 and Figure 2 , Figure 1 This is a schematic diagram of the structures of the MICA and MICB genes. Figure 2 This is a flowchart of the method for high-resolution genotyping of the MICA & MICB genes according to the present invention.

[0049] The method for high-resolution genotyping of the MICA & MICB genes specifically includes the following steps:

[0050] (1) Specific primer set for high-resolution genotyping of MICA & MICB genes

[0051] Forward primer MICA-F1:

[0052] 5'-CCGTGCTTATGAAGTTGGAGCTG-3'(SEQ ID NO.1)

[0053] Reverse primer MICA-R1:

[0054] 5'-ACAGACCTCTCTTTCCCCTGAACC-3'(SEQ ID NO.2)

[0055] Forward primer MICB-F1: 5'-AAAAGGGAGTCTGGAGAGAGCTTGGTGTTG-3' (SEQ ID NO.3)

[0056] Reverse primer MICB-R1:

[0057] 5'-GCTGGTCAGGAGGAGTCATCACCAAGATAC-3'(SEQ ID NO.4)

[0058] (2) Multiplex amplification of MICA & MICB genes

[0059] For long-fragment amplification, TAKARA's Tks Gflex DNA Polymerase was selected. The reagent was thawed on an ice plate, mixed thoroughly, and then microcentrifuged before PCR amplification to obtain the amplified products of the MICA & MICB genes. The PCR amplification reaction program is shown in Table 1.1. The reaction program is as follows:

[0060] Table 1.1 PCR amplification reaction procedure

[0061]

[0062] The PCR amplification reaction system is shown in Table 1.2. Reaction system:

[0063] Table 1.2 PCR amplification reaction system

[0064]

[0065] The primer set mixture was prepared by mixing the four primers in equal proportions (MICA-F1 / MICA-R1 / MICB-F1 / MICB-R1), then diluting it to 0.83 μM with ddH2O or TAE buffer, and then taking 6 μL.

[0066] (3) Purification of amplification products

[0067] The amplification product was purified using magnetic bead purification, and the specific procedure can be found in the conventional magnetic bead purification process in this field.

[0068] (4) Library Construction

[0069] The library construction stage can utilize commercially available universal DNA library construction kits, including but not limited to the following: Qiagen's QIAseq FX DNA Library Kit, Roche's KAPA Hyperplus Kits, and Zekkai's ModμLar DNA Library Kit. The following example uses the QIAseq FX DNA Library Kit.

[0070] The amplification and purification product from step (2) was digested into DNA fragments with a main band of 200-300 bp. After end repair and addition of A bases to the DNA fragments, specific adapters were ligated, and the fragments were purified to construct a library.

[0071] a. Enzyme digestion, end repair, and addition of the A base were carried out according to the reaction system in Table 1.3 and the following reaction procedure: 4℃, 1 min; 32℃, 18 min; 65℃, 30 min; 4℃, ∞.

[0072] Table 1.3 Reaction system for enzyme digestion, end repair, and A base.

[0073]

[0074] b. Connect and purify the reaction product from step a above, and carry out the reaction according to the reaction system in Table 1.4 and the following reaction procedure: 20℃, 15 min; 4℃, ∞.

[0075] Table 1.4 Joint Connection Reaction System

[0076]

[0077] c. The product is purified using magnetic bead purification, and the specific procedure can be found in the conventional magnetic bead purification process in this field.

[0078] d. Use Qubit to determine the library concentration, which should be ≥1 ng / μL; use an Agilent 2100 bioanalyzer (5067-4626 High Sensitivity DNA Kit) to determine the library fragment size, with the main peak of the library fragment at 300-500 bp.

[0079] (5) Sequencing

[0080] The sequencing library was sequenced using the Miseq sequencer on the Illumina platform, with a sequencing length of 2*150 bp.

[0081] (6) Perform bioinformatics analysis on the sequencing data. The flowchart is as follows: Figure 3 As shown, it includes the following steps:

[0082] Step 1: Raw data conversion and preprocessing. The raw data (bcl file) obtained from sequencing is converted into a fastq file after being processed by bcl2fastq. The adapter sequence and low-quality reads (terminal base quality <20) are removed by the fastp tool.

[0083] The second step is data volume reduction. The fastq files processed in the first step are randomly sampled with 1,200,000 reads per sample to generate new fastq files. By using the data volume reduction strategy, the analysis speed can be greatly improved while ensuring the accuracy of the analysis.

[0084] Step 3: HLA reference sequence alignment, using the International Immunogenetic Data (IMGT) database.

[0085] Step 4: Dynamic Depth Adjustment. Since sequencing experiments can lead to localized areas with insufficient coverage depth, causing typing difficulties or errors, this step involves four stages: low-depth site selection and index construction, dynamic data extraction, optimized alignment and filtering, and depth supplementation verification. The specific process is as follows:

[0086] 4.1 Low-depth site screening and core adjustment region construction

[0087] The effective coverage depth of each site was calculated using the Samtools depth tool (effective reads are defined as: MAPQ≥30, mismatches≤2, and no insertions or deletions). Sites with a depth <250X were selected as target optimization sites. For each target site, 50bp of reference sequences upstream and downstream were extracted as core adjustment sequences (101bp).

[0088] 4.2 Dynamic Extraction of Raw Data

[0089] Using the core adjusted sequence reference sequence of the target site, homology retrieval is performed in the original FASTQ file using the BLASTN algorithm, with the alignment threshold set as: sequence matching degree ≥ 85%; at the same time, the pairing relationship of the two-end reads is utilized, if one of the reads matches the core sequence, both reads are extracted simultaneously to ensure the integrity of the read information; after extraction, a dedicated FASTQ file for each target site is generated.

[0090] 4.3 Optimize comparison and filtering

[0091] For each target locus, candidate FASTQ sequences were re-aligned to the IMGT database, and the alignment start position, mismatch sites, and match scores were recorded. Reads meeting the following criteria were selected: ① read alignment quality MAPQ ≥ 30; ② mismatch sites were not located in HLA antigen-binding regions (e.g., exon 2-3 regions of HLA-I genes); ③ reads covered the target locus. The selected valid alignment results were then categorized according to the target locus and a BAM format file was generated.

[0092] 4.4 In-depth supplementary inspection

[0093] The effective coverage depth of each target site after realignment is calculated using the Samtools depth tool. If the depth after enhancement is still <250X, return to step 4.1, expand the retrieval range of the core adjustment sequence (e.g., extend it to 150bp), and re-extract the original data until the target site depth reaches more than 250X.

[0094] Step 5: HLA genotyping. First, HLA-HD analysis software is used for preliminary HLA genotyping. Then, a self-developed script is run to optimize the HLA genotyping results, including extracting the results of the required sites, extracting G group information from the intermediate file, outputting only 4 positions from the mic and 6 positions from the remaining genes, etc. Finally, the genotyping result file in the corresponding format is automatically generated.

[0095] Step 6: Quality control results acquisition. Sort the BAM files and generate an index. Perform statistical analysis on target region coverage, sequencing depth, and insert fragment distribution. Generate corresponding quality control files and import them into IGV for display.

[0096] Example 2: Specific primer set for multiplex amplification of MICA & MICB genes

[0097] In this embodiment, referring to the FASTA sequences related to the MICA & MICB genes in the IMGT / HLA database, primers were designed in the conserved regions flanking the MICA & MICB genes. Three sets of primers were designed by adjusting parameters such as TM, GC, and length: primer set A, primer set B, and primer set C. Specific forward primers MICA-F1 / 2 / 3 were designed upstream of the MICA gene, MICB-F1 / 2 / 3 upstream of the MICB gene, and specific reverse primers MICA-R1 / 2 / 3 downstream of the MICA gene and MICB-R1 / 2 / 3 downstream of the MICB gene. TAKARA's Tks Gflex DNA Polymerase was selected as the amplification enzyme. The specific primer sequences are shown in Table 2.1.

[0098] Table 2.1 Different primer sets for amplifying the MICA & MICB genes

[0099]

[0100] Using these three sets of primers, the target region was amplified according to the method of step (2) in Example 1, and verified by agarose gel electrophoresis.

[0101] The results are as follows Figure 4 As shown, primer set A amplified a clear target fragment, primer set B failed to amplify the target fragment, and primer set C only amplified a small amount of the target fragment. Therefore, primer set A was selected for subsequent amplification and detection of the MICA and MICB genes.

[0102] Example 3: Optimization of enzyme reagents for high-resolution genotyping of MICA & MICB genes.

[0103] This embodiment follows the method for high-resolution genotyping of MICA & MICB genes in Example 1, and optimizes the amplification enzyme reagents, specifically including:

[0104] Five DNA samples (one human genome standard NA12878+ and four clinical samples with known results) were tested using different Taq enzymes and different reaction procedures.

[0105] Reagent A: Tks Gflex DNA Polymerase, Manufacturer: TAKARA;

[0106] Reagent B: 2 × Vazyme LAmp® Master Mix, Manufacturer: Novizan;

[0107] Reagent C: KAPA2G Fast Multiplex PCR Kit (Manufacturer: KAPA BIOSYSTEMS).

[0108] Primers used were primer set A, and the working concentration of the primer mixture was 0.83 μM. Details are shown in Table 3.1.

[0109] Table 3.1 Genotyping results of MICA & MICB obtained by amplification with different Taq enzymes

[0110]

[0111] Amplification was performed using the enzyme reagents listed in Table 3.1. The amplified products were then subjected to library construction, sequencing, and bioinformatics analysis to obtain high-resolution genotyping of the MICA and MICB genes. The results are shown in Table 3.1. When using Tks Gflex DNA Polymerase, correct genotyping was detected in all 5 samples, indicating that this enzyme meets the requirements for long-fragment multiplex PCR in this invention. When using 2 × Vazyme LAmp® Master Mix, the MICB gene genotyping of samples NA12878 and MIC-4 was not detected, and the MICB gene genotyping of sample MIC-2 was incorrect, suggesting that this enzyme may not be suitable for multiplex PCR. When using the KAPA2G Fast Multiplex PCR Kit, correct genotyping was not detected in any of the 5 samples, suggesting that this enzyme is not suitable for amplifying long-fragment PCR. Considering the performance ratio, Tks Gflex DNA Polymerase was selected as the amplification reagent for the subsequent MICA and MICB genes.

[0112] Example 4: Primer concentration optimization for multiplex amplification of MICA & MICB genes

[0113] This embodiment follows the method for high-resolution genotyping of MICA & MICB genes in Example 1 to explore the optimal primer concentration, specifically including the following steps:

[0114] (1) First, mix primer set A in equal proportion (i.e., 4 primers in equal proportion), and then dilute to 0.083 μM, 0.83 μM and 8.3 μM respectively to prepare working primer mixtures for later use;

[0115] (2) The MICA & MICB genes were amplified using the human genome standard NA12878 sample and the enzyme reagent Tks Gflex DNA Polymerase, and the concentration of the amplification products was determined.

[0116] (3) After constructing the library, the size of the library fragments was determined using an Agilent 2100 bioanalyzer.

[0117] Table 3 Concentration of amplification products

[0118]

[0119] The results are as follows Figure 5 As shown in Table 3, Figure 5 In the table, A, B, and C represent the library fragment sizes after testing with the human genome standard NA12878 using primer mixtures of 0.083 / 0.83 / 8.3 μM, respectively. Figure 5 As shown in Table 3, the target fragment (300-500 bp) was not amplified at 0.083 μM; amplification was successful at 0.83 μM and the target fragment (300-500 bp) could be detected. However, non-target fragments were present at 8.3 μM. It is speculated that the increase in primer concentration led to the formation of primer dimers. Therefore, a mixed primer with a concentration of 0.83 μM was selected for the subsequent amplification of the MICA and MICB genes.

[0120] Example 5: Methodological Validation of the Method of the Invention

[0121] The methodology of the detection method of the present invention was validated using the method in Example 1 through 16 DNA samples (1 NTC sample + 1 human genome standard NA12878 sample + 14 clinical samples with known results).

[0122] Table 5.1 Comparison of Methodological Validation Results of the Invention

[0123]

[0124]

[0125]

[0126] The results are shown in Table 5.1. The validation results of 16 validation samples were compared with known results, and the results were completely consistent, indicating that the accuracy of the high-resolution genotyping detection of MICA & MICB genes was 100%. It is suitable for large-scale matching and gene screening, and is applicable to matching for organ and bone marrow transplantation and screening for susceptibility genes in autoimmune diseases.

[0127] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A primer for detecting high-resolution typing of MICA and MICB genes, characterized by, The primers include: Primer pairs targeting the MICA gene: SEQ ID NO:1 and SEQ ID NO:2; Primer pairs SEQ ID NO:3 and SEQ ID NO:4 targeting the MICB gene.

2. The application of the primers for high-resolution genotyping of MICA and MICB genes as described in claim 1 in the detection of high-resolution genotyping of MICA and MICB genes.

3. A kit for detecting high-resolution typing of MICA and MICB genes, characterized by, The kit includes the primers as described in claim 1.

4. The kit of claim 3, wherein The kit also includes PCR amplification reagents; the PCR amplification reagents include DNA polymerase and PCR buffer. Preferably, the PCR amplification reagent includes Tks Gflex DNA Polymerase; and / or the PCR buffer is 2x Gflex PCR Buffer containing Mg 2+ and dNTPs.

5. The use of the kit for high-resolution genotyping of MICA and MICB genes according to any one of claims 3-4 in the detection of high-resolution genotyping of MICA and MICB genes.

6. A method for detecting high-resolution typing of MICA and MICB genes, characterized by, Includes the following steps: Step 1. Mix the primer pair targeting the MICA gene and the primer pair targeting the MICB gene to obtain mixed primers; the primer pair sequences targeting the MICA gene are shown in SEQ ID NO:1 and SEQ ID NO:2, and the primer pair sequences targeting the MICB gene are shown in SEQ ID NO:3 and SEQ ID NO:

4. Step 2. Using the DNA sample to be tested as a template and the mixed primers from Step 1 as primers, perform PCR amplification to obtain the amplification product, and purify the obtained amplification product; Step 3. Perform enzyme digestion, end repair, A base addition, and adapter ligation on the amplification products to construct a sequencing library; Step 4. Sequencing the sequencing library.

7. The detection method as described in claim 6, characterized in that, The working concentration of the primers is 0.5 μM to 5 μM.

8. The detection method as described in claim 7, characterized in that, The working concentration of the primers is 0.5 μM to 1 μM, preferably 0.7 μM to 0.9 μM.

9. The detection method as described in claim 6, characterized in that, In step 1, the primers in the mixed primers are mixed in equal proportions; And / or, in step 3, the sequencing is performed using the Illumina sequencing platform; And / or, in step 4, the library concentration is ≥1 ng / μL.

10. The detection method as described in claim 6, characterized in that, In step 2, the PCR amplification reaction procedure is as follows: Pre-denaturation: Treat at 93℃~95℃ for 1min~2min, 1 cycle; Amplification cycle: 95℃~100℃ for 8 sec~12 sec, 65℃~70℃ for 4 min~6 min, for a total of 25 cycles; And / or, the amplification reaction system is as follows: a 25 μL reaction system containing 90 ng to 110 ng of DNA template, 0.5 to 1.5 μL of Tks Gflex DNA Polymerase, 10 to 15 μL of 2×Gflex PCR Buffer, 3 to 8 μL of mixed primers, and the remainder being water.

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