MICB gene whole exon PCR-SBT typing kit and application thereof

Abstract

The invention provides an MICB gene whole exon PCR-SBT typing kit and application thereof, and relates to the technical field of biological medicine. According to the kit, six exons of the MICB gene are analyzed by providing three pairs of amplification primers and four sequencing primers with good specificity, so that typing of the MICB gene is realized, the problems that typing of the MICB gene is ambiguous and specific alleles cannot be accurately judged in the prior art can be solved, and application of the MICB gene in clinic and scientific research is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a MICB gene whole exon PCR-SBT typing kit and application thereof. Background Art

[0002] The major histocompatibility complex class Ⅰ chain-related gene B (MICB) belongs to the non-classical human leukocyte antigen (HLA) class Ⅰ gene. The MICB protein it encodes is one of the ligands of NKG2D. It can activate NK cells, CD8 αβ T cells, γδ T cells, etc. that express NKG2D on the cell surface, providing a molecular basis for these cells to exert their innate immune function.

[0003] The MICB gene is located on the short arm of chromosome 6, is about 12.9 kb long, and is highly polymorphic. Currently, there are 295 alleles in the HLA database. Studies have shown that MICB gene polymorphism is associated with susceptibility to diseases such as lung cancer, breast cancer, leukemia M, rheumatoid arthritis, and ulcerative colitis. Some studies also believe that MICB gene polymorphism affects the prognosis of hematopoietic stem cell transplantation, so MICB gene typing before transplantation is beneficial to the long-term survival of patients. Therefore, it is necessary to establish an accurate and low-cost detection method for the MICB gene.

[0004] At present, the main MICB genotyping technologies include polymerase chain reaction-sequence specific primer (PCR-SSP), polymerase chain reaction-sequence based typing (PCR-SBT) and next generation sequencing (NGS). PCR-SSP has the advantage of rapid MICB genotyping, but because its primer design requires the sequence information of the target site in advance, it may not be able to identify newly emerged variants. In addition, because PCR-SSP requires 29 PCR reactions and then gel electrophoresis analysis for MICB genotyping, the technical differences of operators and experimental operation errors may affect the judgment of the results. The emergence of NGS provides convenience for high-throughput detection and can discover new alleles, but its data processing complexity, error rate and high cost limit its application.

[0005] PCR-SBT is a high-resolution HLA genotyping technology recommended by the World Health Organization. It can directly obtain DNA sequences and identify all existing polymorphisms (including known and new variations). It is the most direct and intuitive typing method. Therefore, there are many studies on MICB genotyping using PCR-SBT, but sometimes ambiguous results may occur, which is mainly related to insufficient number of exons detected and unreasonable primer design. For example, the MICB gene has a total of 6 exons, but the current PCR-SBT typing of the MICB gene mainly analyzes its exons 2-5. There are also polymorphic sites in exon 1 and exon 6, and they are polymorphic sites that are necessary for distinguishing certain alleles. For example, the difference between MICB*002:01 and MICB*002:06 is only in exon 1, and the difference between MICB*005:02, MICB*005:13, and MICB*005:14 is also only in exon 1; the difference between MICB*005:02, MICB*010, and MICB*005:08 is only in exon 6; the difference between MICB*004:01 and MICB*028, and between MICB*019:01 and MICB*038 is also only in exon 6. Among all the alleles of the MICB gene, MICB*005:02 is the most common allele, MICB*002:01 and MICB*004:01 are also relatively common alleles, and MICB*010, MICB*005:08, MICB*028, and MICB*019:01 have all been reported in the Chinese population. Therefore, in order to distinguish these alleles, it is necessary to analyze exons 1 and 6 as well. In addition, due to the high similarity between the MICB gene sequence and the MICA gene sequence, when designing the amplification primers for the MICB gene, the primers must only amplify the MICB gene, but not the MICA gene. If the specificity of the primers is not considered during primer design, the MICA gene may be amplified while the MICB gene is amplified, resulting in ambiguous results.

[0006] Therefore, it is urgent to provide a solution to solve the above problems. Summary of the invention

[0007] The present invention aims to provide a MICB gene whole exon PCR-SBT typing kit and its application, which can analyze the six exons of the MICB gene and realize the typing of the MICB gene, can improve the problem of ambiguity in the typing of the MICB gene and inability to accurately determine the specific allele in the prior art, and is conducive to the application of the MICB gene in clinical and scientific research.

[0008] In a first aspect, the present invention provides a MICB gene whole exon PCR-SBT typing kit, comprising 3 pairs of amplification primers and 4 sequencing primers; The base sequence of amplification primer pair 1 is shown in SEQ ID NO. 1-2; The base sequence of amplification primer pair 2 is shown in SEQ ID NO.3-4; The base sequence of amplification primer pair 3 is shown in SEQ ID NO.5-6; Sequencing primer 1 is the upstream primer of amplification primer pair 1, and the base sequence is shown in SEQ ID NO.1; Sequencing primer 2 is the upstream primer of amplification primer pair 2, and the base sequence is shown in SEQ ID NO.3; The base sequence of sequencing primer 3 is shown in SEQ ID NO.7; Sequencing primer 4 is the upstream primer of amplification primer pair 3, and the base sequence is shown in SEQ ID NO.5.

[0009] Specifically, the amplification primer pair 1 is an amplification primer for exon 1; The amplification primer pair 2 is an amplification primer for exons 2-5; The amplification primer pair 3 is an amplification primer for exon 6; The sequencing primer 1 is a sequencing primer for exon 1; The sequencing primer 2 is a sequencing primer for exon 2 and exon 3; The sequencing primer 3 is a sequencing primer for exon 4 and exon 5; The sequencing primer 4 is a sequencing primer for exon 6.

[0010] In a second aspect, the present invention provides an application of a MICB gene whole exon PCR-SBT typing kit in detecting MICB genotyping, comprising the following steps: S1. Extract sample genomic DNA and dilute to 10 ng / μL; S2, using the three pairs of amplification primers to amplify exons 1-6 of the MICB gene respectively to obtain amplified products; S3, purifying the amplified product to obtain a purified product; S4, using the sequencing primers to sequence the purified products respectively to obtain sequencing results; S5. Compare and analyze the sequencing results with the sequences of each allele of the MICB gene to obtain the scoring results.

[0011] Specifically, when amplifying exon 1 in step S2, the following steps are included: A 10 μL reaction system was prepared, including 5 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 1, 0.4 μmol / L downstream primer of amplification primer pair 1, and 5 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 68°C, 20 s; 72°C, 20 s; 35 cycles; 72°C, 5 min.

[0012] Specifically, when exons 2-5 are amplified in step S2, the following steps are included: Prepare a 20 μL reaction system, including 10 μL 2× PrimeSTAR Max Premix, 0.4 μmol / L upstream primer of amplification primer pair 2, 0.4 μmol / L downstream primer of amplification primer pair 2, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction program: 98 °C, 10 s; 66 °C, 5 s; 72 °C, 11 s; 35 cycles; 72 °C, 5 min.

[0013] Specifically, when exon 6 is amplified in step S2, the following steps are included: A 20 μL reaction system was prepared, including 10 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 3, 0.4 μmol / L downstream primer of amplification primer pair 3, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 63°C, 20 s; 72°C, 30 s; 35 cycles; 72°C, 5 min.

[0014] The beneficial effects of the present invention include: (1) The kit provided by the present invention can realize MICB genotyping by analyzing the six exons of the MICB gene, and can identify all polymorphic sites on the six exons, thereby avoiding misjudgment of alleles caused by the lack of analysis of all exons; (2) The MICB gene whole exon PCR-SBT typing kit provided by the present invention has low cost and is easy to operate, while the typing time is short and the typing results are accurate, which is conducive to the application of MICB gene typing in clinical and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0016] Figure 1 The electrophoresis result of the PCR product of exon 1 of the sample in Example 2; Figure 2 The electrophoresis results of the PCR products of exons 2-5 of the sample in Example 2; Figure 3 The electrophoresis result of the PCR product of exon 6 of the sample in Example 2; Figure 4 This is a diagram showing the sequencing results of exon 1 of the sample in Example 2; Figure 5 This is a diagram of the sequencing results of exon 2 of the sample in Example 2; Figure 6 This is a diagram showing the sequencing results of exon 3 of the sample in Example 2; Figure 7 This is a diagram showing the sequencing results of exon 4 of the sample in Example 2; Figure 8 This is a diagram showing the sequencing results of exon 5 of the sample in Example 2; Fig. 9 This is a diagram showing the sequencing results of exon 6 of the sample in Example 2. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described through the following embodiments in conjunction with the accompanying drawings. However, the following embodiments are illustrative and are only used to specifically describe the present invention rather than to limit the present invention.

[0018] The present invention provides a MICB gene whole exon PCR-SBT typing kit, comprising 3 pairs of amplification primers and 4 sequencing primers; The base sequence of amplification primer pair 1 is shown in SEQ ID NO. 1-2; The base sequence of amplification primer pair 2 is shown in SEQ ID NO.3-4; The base sequence of amplification primer pair 3 is shown in SEQ ID NO.5-6; Sequencing primer 1 is the upstream primer of amplification primer pair 1, and the base sequence is shown in SEQ ID NO.1; Sequencing primer 2 is the upstream primer of amplification primer pair 2, and the base sequence is shown in SEQ ID NO.3; The base sequence of sequencing primer 3 is shown in SEQ ID NO.7; Sequencing primer 4 is the upstream primer of amplification primer pair 3, and the base sequence is shown in SEQ ID NO.5.

[0019] Specifically, the amplification primer pair 1 is an amplification primer for exon 1; The amplification primer pair 2 is an amplification primer for exons 2-5; The amplification primer pair 3 is an amplification primer for exon 6; The sequencing primer 1 is a sequencing primer for exon 1; The sequencing primer 2 is a sequencing primer for exon 2 and exon 3; The sequencing primer 3 is a sequencing primer for exon 4 and exon 5; The sequencing primer 4 is a sequencing primer for exon 6.

[0020] The present invention also provides an application of a MICB gene all-exon PCR-SBT typing kit in detecting MICB genotyping, comprising the following steps: S1. Extract sample genomic DNA and dilute to 10 ng / μL; S2, using the three pairs of amplification primers to amplify exons 1-6 of the MICB gene respectively to obtain amplified products; S3, purifying the amplified product to obtain a purified product; S4, using the sequencing primers to sequence the purified products respectively to obtain sequencing results; S5. Compare and analyze the sequencing results with the sequences of each allele of the MICB gene to obtain the scoring results.

[0021] Specifically, when amplifying exon 1 in step S2, the following steps are included: A 10 μL reaction system was prepared, including 5 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 1, 0.4 μmol / L downstream primer of amplification primer pair 1, and 5 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 68°C, 20 s; 72°C, 20 s; 35 cycles; 72°C, 5 min.

[0022] Specifically, when exons 2-5 are amplified in step S2, the following steps are included: Prepare a 20 μL reaction system, including 10 μL 2× PrimeSTAR Max Premix, 0.4 μmol / L upstream primer of amplification primer pair 2, 0.4 μmol / L downstream primer of amplification primer pair 2, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction program: 98 °C, 10 s; 66 °C, 5 s; 72 °C, 11 s; 35 cycles; 72 °C, 5 min.

[0023] Specifically, when exon 6 is amplified in step S2, the following steps are included: A 20 μL reaction system was prepared, including 10 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 3, 0.4 μmol / L downstream primer of amplification primer pair 3, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 63°C, 20 s; 72°C, 30 s; 35 cycles; 72°C, 5 min.

[0024] In some embodiments, the method for designing primers in the MICB gene all-exon PCR-SBT typing kit comprises the following steps: D1. Determine the location of the exon in the DNA sequence of the MICB gene; D2, downloading and saving the sequences of all alleles of the MICB gene in a database, including the sequences of 5'UTR, exons, introns, 3'UTR and CDS; D3. Use oligo 7 to amplify each exon of the MICB gene and design sequencing primers. Each primer is required to have no mismatch in all alleles of the MICB gene and be different from the MICA gene sequence. The designed primer sequences are shown in SEQ ID NO.1-7.

[0025] Example 1

[0026] Embodiment 1 of the present invention provides a method for designing primers in a MICB gene all-exon PCR-SBT typing kit, comprising the following steps: D1. Download the DNA sequence of the MICB gene from the NCBI website (https: / / www.ncbi.nlm.nih.gov / gene / ) and identify the locations of the six exons in the gene; D2. Download and save the sequences of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, 5'UTR, intron 1, intron 5, 3'UTR, and CDS of all alleles of the MICB gene from the IMGT / HLA database (https: / / www.ebi.ac.uk / ipd / imgt / hla / alignment / ); D3. Use oligo 7 to amplify each exon of the MICB gene and design sequencing primers. Each primer is required to have no mismatch in all alleles of the MICB gene and be different from the MICA gene sequence. The designed primer sequences are shown in Table 1. Wherein, amplification primer pair 1 includes MICB-F1 and MICB-R1, which are primers for amplification of exon 1, and the sequence is shown in SEQ ID NO.1-2; Amplification primer pair 2 includes MICB-F2 and MICB-R2, which are primers for amplification of exons 2-5, and the sequences are shown in SEQ ID NO. 3-4; Amplification primer pair 3 includes MICB-F3 and MICB-R3, which are primers for amplification of exon 6, and the sequences are shown in SEQ ID NO.5-6; Sequencing primer 1 is MICB-F1, i.e., the upstream primer of amplification primer pair 1, which is the sequencing primer of exon 1, and its sequence is shown in SEQ ID NO.1; Sequencing primer 2 is MICB-F2, i.e., the upstream primer of amplification primer pair 2, which is a sequencing primer for exon 2 and exon 3, and its sequence is shown in SEQ ID NO. 3; Sequencing primer 3 is MICB-F4, which is a sequencing primer for exon 4 and exon 5, and the sequence is shown in SEQ ID NO.7; Sequencing primer 4 is MICB-F3, ie, the upstream primer of amplification primer pair 3, and is the sequencing primer of exon 6, and its sequence is shown in SEQ ID NO.5.

[0027] Table 1 Sequences of primers designed in Example 1

[0028]

[0029] Example 2

[0030] Embodiment 2 of the present invention provides an application of a MICB gene whole exon PCR-SBT typing kit in detecting MICB genotyping, comprising the following steps: S1. Use AxyPrep Blood Genomic DNA Mini Kit to extract genomic DNA from blood samples. After quantifying the extracted genomic DNA using a micro-nucleic acid protein quantifier, take a portion of the sample and dilute it to 10 ng / μl as a PCR template. S2. Since intron 1 and intron 5 of the MICB gene are relatively large, while intron 2, intron 3, and intron 4 are relatively small, the six exons are divided into three fragments for amplification, wherein exon 1 and exon 6 are amplified separately, and exon 2 to exon 5 are amplified together, including the following sub-steps; S2.1. Amplification of exon 1

[0031] A 10 μL reaction system was prepared, including 5 μL 2× Taq Master Mix (Suzhou Nearshore Protein Technology Co., Ltd.), 0.4 μmol / L MICB-F1, 0.4 μmol / L MICB-R1, and 5 ng genomic DNA; After preparation, PCR amplification was performed, and the reaction program was as follows: 94 °C, 3 min; 94 °C, 20 s; 68 °C, 20 s; 72 °C, 20 s; 35 cycles; 72 °C, 5 min; S2.2, amplification of exon 2, exon 3, exon 4, and exon 5

[0032] A 20 μL reaction system was prepared, including 10 μL 2× PrimeSTAR Max Premix (TaKaRa), 0.4 μmol / L MICB-F2, 0.4 μmol / L MICB-R2, and 10 ng genomic DNA; After preparation, PCR amplification was performed, and the reaction program was: 98 °C, 10 s; 66 °C, 5 s; 72 °C, 11 s; 35 cycles; 72 °C, 5 min; S2.3. Amplification of exon 6

[0033] A 20 μL reaction system was prepared, including 10 μL 2× Taq Master Mix (Suzhou Nearshore Protein Technology Co., Ltd.), 0.4 μmol / L MICB-F3, 0.4 μmol / L MICB-R3, and 10 ng genomic DNA; After preparation, PCR amplification was performed, and the reaction program was as follows: 94 °C, 3 min; 94 °C, 20 s; 63 °C, 20 s; 72 °C, 30 s; 35 cycles; 72 °C, 5 min; The electrophoresis results of the PCR amplification products of exon 1 are as follows Figure 1 As shown, lanes 1-3 are PCR products of 3 test samples, lane 4 is a negative control, and lane 5 is Trans DNA Marker I (Beijing Quanshijin Biotechnology Co., Ltd., from top to bottom: 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp); The electrophoresis results of the PCR amplification products of exons 2-5 are as follows Figure 2As shown, lanes 1-3 are PCR products of 3 test samples, lane 4 is a negative control, and lane 5 is a 1Kb DNA ladder (Beijing Quanshijin Biotechnology Co., Ltd., from top to bottom: 10000 bp, 8000 bp, 6000 bp, 5000 bp, 4000 bp, 3000 bp, 2000 bp, 1000 bp); The electrophoresis results of the PCR amplification products of exon 6 are as follows Figure 3 As shown, lanes 1-3 are PCR products of 3 test samples, lane 4 is a negative control, and lane 5 is a 100 bp DNA Ladder (Beijing Quanshijin Biotechnology Co., Ltd., from top to bottom: 1500 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp); S3, purifying the exon amplification product in step S2 using a gel excision recovery method; S4. Use BigDye Terminator Cycle Sequencing Kit (ABI, USA) to amplify the purified product with sequencing primers, precipitate the amplified product with isopropanol, load it into DNA Analyzer 3730XL sequencer for sequence analysis, and obtain the sequencing map; S5, using Chromas software to interpret the sequencing map obtained in step S4, aligning it with the sequences of each allele of the MICB gene, and finally determining the sample allele; The sequencing results of exons 1-6 can be found in Figures 4 to 9 , the results showed that the two alleles of this sample were MICB*005:02 and MICB*014:02.

[0034] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A MICB gene whole exon PCR-SBT typing kit, characterized in that: Includes 3 pairs of amplification primers and 4 sequencing primers; The base sequence of amplification primer pair 1 is shown in SEQ ID NO. 1-2; The base sequence of amplification primer pair 2 is shown in SEQ ID NO.3-4; The base sequence of amplification primer pair 3 is shown in SEQ ID NO.5-6; Sequencing primer 1 is the upstream primer of amplification primer pair 1, and the base sequence is shown in SEQ ID NO.1; Sequencing primer 2 is the upstream primer of amplification primer pair 2, and the base sequence is shown in SEQ ID NO.3; The base sequence of sequencing primer 3 is shown in SEQ ID NO.7; Sequencing primer 4 is the upstream primer of amplification primer pair 3, and the base sequence is shown in SEQ ID NO.

5.

2. The kit according to claim 1, characterized in that The amplification primer pair 1 is an amplification primer for exon 1; The amplification primer pair 2 is an amplification primer for exons 2-5; The amplification primer pair 3 is an amplification primer for exon 6; The sequencing primer 1 is a sequencing primer for exon 1; The sequencing primer 2 is a sequencing primer for exon 2 and exon 3; The sequencing primer 3 is a sequencing primer for exon 4 and exon 5; The sequencing primer 4 is a sequencing primer for exon 6.

3. Use of the kit according to any one of claims 1 to 2 in detecting MICB genotyping.

4. The use according to claim 3, characterized in that: The following steps are involved: S1. Extract sample genomic DNA and dilute to 10 ng / μL; S2, using the three pairs of amplification primers to amplify exons 1-6 of the MICB gene respectively to obtain amplified products; S3, purifying the amplified product to obtain a purified product; S4, using the sequencing primers to sequence the purified products respectively to obtain sequencing results; S5. Compare and analyze the sequencing results with the sequences of each allele of the MICB gene to obtain the scoring results.

5. The use according to claim 4, characterized in that: When amplifying exon 1 in step S2, the following steps are included: A 10 μL reaction system was prepared, including 5 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 1, 0.4 μmol / L downstream primer of amplification primer pair 1, and 5 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 68°C, 20 s; 72°C, 20 s; 35 cycles; 72°C, 5 min.

6. The use according to claim 4, characterized in that: When amplifying exons 2-5 in step S2, the following steps are included: Prepare a 20 μL reaction system, including 10 μL 2× PrimeSTAR Max Premix, 0.4 μmol / L upstream primer of amplification primer pair 2, 0.4 μmol / L downstream primer of amplification primer pair 2, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction program: 98 °C, 10 s; 66 °C, 5 s; 72 °C, 11 s; 35 cycles; 72 °C, 5 min.

7. The use according to claim 4, characterized in that: When amplifying exon 6 in step S2, the following steps are included: A 20 μL reaction system was prepared, including 10 μL 2× Taq Master Mix, 0.4 μmol / L upstream primer of amplification primer pair 3, 0.4 μmol / L downstream primer of amplification primer pair 3, and 10 ng genomic DNA; After preparation, PCR amplification was performed with the following reaction procedure: 94°C, 3 min; 94°C, 20 s; 63°C, 20 s; 72°C, 30 s; 35 cycles; 72°C, 5 min.

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