CN111334578A - IGHMBP2 gene mutation site detection kit and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and relates to a detection kit for IGHMBP2 gene mutation sites and application thereof. The detection kit comprises an amplification primer and an extension primer, wherein the sequence of a forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of a reverse primer of the amplification primer is shown as SEQ ID NO.4, the sequence of the extension primer is shown as SEQ ID NO.5, the sequence of the IGHMBP2 gene is shown as SEQ ID NO.1, and the sequence of the mutated IGHMBP2 gene is shown as SEQ ID NO. 2. The IGHMBP2 gene mutation site detection kit can predict the radiation sensitivity of personnel with high efficiency and low cost.

Description

IGHMBP2 gene mutation site detection kit and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and relates to a detection kit for IGHMBP2 gene mutation sites and application thereof.

Background

Currently, radiation therapy remains the mainstay of modern cancer therapy, and approximately 50% of cancer patients require radiation therapy. However, patients receiving the same dose of radiation therapy exhibit different radiotoxicity: few have no obvious toxic effects, most have mild or moderate toxic reactions clinically, while few cause severe normal tissue complications and may even be life threatening. Therefore, there is a need to find a molecular marker associated with radiosensitivity for predicting whether a patient is a radiosensitive (or radioresistant) individual.

The prediction of radiation sensitivity is to be able to tailor the radiation treatment regimen to individual patients to improve prognosis, so that on the one hand the radiation dose can be reduced to reduce toxicity to sensitive individuals, and on the other hand the radiation dose can be increased to allow more radiation resistant patients to be effectively treated. This will maximize control of the tumor while minimizing damage to the patient's normal tissues. In addition, about 20 thousands of radiation workers exist in China, the radiation workers need to receive professional irradiation for a long time during daily work, and prediction of radiation sensitivity of the radiation workers is helpful for preventing and reducing professional injuries.

The site IGHMBP2 rs1249463, at chromosome 11, 68904009, is located at the allele T > C, and is a gene encoding a member of the helicase superfamily that binds to specific DNA sequences of the immunoglobulin mu chain switch region. Mutations in this gene can lead to spinal muscular atrophy with respiratory distress type I.

Single nucleotide polymorphism markers (SNPs) are "third generation DNA genetic markers", and 300 ten thousand of them exist in the human genome, and are considered as genetic markers with the best application prospect. SNPs can truly reflect genetic differences and are associated with radiation-induced toxicity in normal tissues.

In addition, compared with the traditional gene detection method mainly based on sequencing and hybridization principles, the matrix-assisted laser desorption ionization mass spectrometry technology has incomparable advantages in the aspects of detection efficiency, detection flux, detection sensitivity, detection accuracy, detection repeatability, detection cost and the like. The technology can realize simultaneous detection of 384 samples, the detection of each detection point only needs 3-5s, pmol level can be detected, and the accuracy reaches more than 98%. Therefore, the matrix-assisted laser desorption ionization mass spectrometry is used for judging the radiation sensitivity, so that the personnel radiation sensitivity can be quickly, accurately, high in flux and low in cost.

Disclosure of Invention

The invention aims to provide a kit for detecting mutation sites of IGHMBP2 gene, which can predict the radiation sensitivity of people with high efficiency and low cost.

To achieve the purpose, in a basic embodiment, the invention provides an IGHMBP2 gene mutation site detection kit, which comprises an amplification primer and an extension primer,

the sequence of the forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of the reverse primer of the amplification primer is shown as SEQ ID NO.4,

the sequence of the extension primer is shown as SEQ ID NO.5,

the sequence of the IGHMBP2 gene is shown as SEQ ID NO.1, the mutation site is rs1249463 site (mutation from T to C) of IGHMBP2 gene, and the sequence of the mutated IGHMBP2 gene is shown as SEQ ID NO. 2.

In a preferred embodiment, the invention provides a kit for detecting mutation sites of IGHMBP2 gene, wherein the concentration of the forward primer of the amplification primer is 2-3 μ M.

In a preferred embodiment, the invention provides a kit for detecting the mutation site of IGHMBP2 gene, wherein the concentration of the reverse primer of the amplification primer is 2-3 μ M.

In a preferred embodiment, the invention provides a kit for detecting the mutation site of IGHMBP2 gene, wherein the concentration of the extension primer is 3.5-4.5. mu.M.

The second purpose of the invention is to provide the application of the detection kit for preparing the kit for predicting the radiation sensitivity of the personnel, so that the radiation sensitivity of the personnel can be predicted with high efficiency and low cost.

To achieve this object, in a basic embodiment, the present invention provides the use of the above-described detection kit for the preparation of a kit for predicting the radiation sensitivity of a person.

The kit for detecting the mutation site of the IGHMBP2 gene has the advantages of high efficiency and low cost and can predict the radiation sensitivity of personnel.

Detailed Description

The following examples further illustrate the practice of the present invention, but the embodiments of the present invention are not limited to the following examples.

Example 1:

1) sample acquisition and irradiation and chromosome aberration analysis

Collecting peripheral blood of healthy adult male of 20-30 years old, and administering 0, 2Gy⁶⁰And (4) irradiating Co gamma rays. And (3) carrying out chromosome aberration analysis on the 2Gy gamma ray irradiation sample, and dividing the population into a susceptible group, a general group and an insensitive group. After the irradiation, the blood sample is cultured for 52h, and then the chromosome is harvested and sliced. Each sample was analyzed for 200 metaphase phases.

2) Peripheral blood genome DNA extraction

Genomic DNA of 0Gy irradiated samples of susceptible and non-susceptible groups was extracted. The whole blood genome DNA extraction is carried out by adopting a blood genome DNA extraction kit of Beijing Tianzhu Biochemical technology Co., Ltd according to a product specification, and the specific steps are shown in the specification. The quantitative detection A260/280 of the sampled nucleic acid is between 1.70 and 1.90, the quality meets the experiment requirements, and the subsequent experiment can be carried out.

3) Whole exon capture sequencing

Sequencing data is firstly subjected to data filtration to remove low-quality data, and clear Reads are obtained. The sequencing needs to reach the clean Reads rate of more than 90%, the clean base rate of more than 20G, the clean base rate of more than 90%, and the Q20 rate of more than 98%, and the experimental sample meets the requirements.

4) Biological information analysis

Clear Reads were aligned to the reference genome and differential SNP sites were screened. Reference genome version: GRCh37(hg19), ftp:// ftp.1000genes.ebi.ac.uk/vol 1/ftp/technical/reference/human _ g1k _ v37. fasta.gz. Differential site IGHMBP2 rs1249463 site was selected by bioinformatics analysis, see Table 1.

TABLE 1 selected radiation sensitive sites

SNP site	Gene	SNP site position	Alleles
				rs1249463	IGHMBP2	chr11：68904009	T/C

Example 2:

1) the method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) human whole blood genome (blood from healthy male volunteers between 20-30 years old) DNA extraction was performed according to the product instructions.

2) The amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 is adopted, and a specific reaction system (5 mu l of the reaction system comprises 0.95 mu l H)₂O、0.625μl PCR Buffer(10×)、0.325μl MgCl₂PCR reaction (25mM), 1. mu.l dNTP (2.5mM), 1. mu.l primer, 0.1. mu.l HotstarTaq (5U/. mu.L)) was performed according to the following reaction program: 15min at 94 ℃; [94 ℃, 20sec, 56 ℃, 30sec ]]45 cycles; 72 ℃ for 4 min. The reaction product was stored at 4 ℃.

3) Using an SAP reaction solution (2. mu.l SAP reaction solution included 1.53. mu.l H)₂O, 0.17. mu.l of SAP Buffer (10 ×), 0.3. mu.l of SAP enzyme (1U/. mu.L)) were applied to the reaction product of step 2) at 37 ℃ for 40min and 85 ℃ for 5min, and the treated product was stored at 4 ℃.

4) Carrying out extension reaction on the treated product in the step 3) by adopting an extension primer of SEQ ID NO.5,

2 μ l reaction included 0.755 μ l H₂O, 0.2. mu.l of iPLex Buffer (10 ×), 0.2. mu.l of iPLEX termination mix, 0.041. mu.l of iPLex enzyme, 0.804. mu.l of primer.

The reaction procedure is as follows: 30s at 94 ℃; 5 cycles of [94 ℃, 5s, (52 ℃, 5s, 80 ℃, 5s) ]40 cycles; 72 ℃ for 3 min. The extension product was stored at 4 ℃.

5) Purifying the extension product of step 4). 6mg of resin was uniformly applied to cover the 384 well plate and left for 20 min. The 384 well plate containing the extension product of step 4) was centrifuged at 1000rpm for 1min, 25. mu.L of deionized water was added to each well, inverted on the resin plate, and then the resin plate was snapped on the 384 well plate in the inverted position, and the resin was dropped into the 384 well plate by tapping, and the membrane was sealed. The 384 well plate was inverted for 20 minutes with the long axis of the 384 well plate as the axis, and centrifuged at 3500rpm for 5 minutes for use.

6) Detecting the genotype of the gene locus: the sample treated in step 5) was transferred to a MassARRAYPectroCHIP chip (SAMSUNG, MassArray. TM. Nanodispenser) and put into a mass spectrometer (SEQUENOM, MassARRAY compact System) for detection.

The detection result shows that the primer designed by the embodiment can detect the rs1249463 locus genotype of the IGHMBP2 gene and can be used for detecting the mutation of the radiation-sensitive gene IGHMBP2 rs1249463 locus.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.

Sequence listing

<110> China institute for radiation protection

<120> IGHMBP2 gene mutation site detection kit and application thereof

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<141>2020-03-24

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Claims (5)

1. A detection kit for mutation sites of IGHMBP2 gene is characterized in that: the detection kit comprises an amplification primer and an extension primer,

the sequence of the forward primer of the amplification primer is shown as SEQ ID NO.3, the sequence of the reverse primer of the amplification primer is shown as SEQ ID NO.4,

the sequence of the extension primer is shown as SEQ ID NO.5,

the sequence of the IGHMBP2 gene is shown as SEQ ID NO.1, and the sequence of the mutated IGHMBP2 gene is shown as SEQ ID NO. 2.

2. The detection kit according to claim 1, characterized in that: the concentration of the forward primer of the amplification primer is 2-3 mu M.

3. The detection kit according to claim 1, characterized in that: the concentration of the reverse primer of the amplification primer is 2-3 mu M.

4. The detection kit according to claim 1, characterized in that: the concentration of the extension primer is 3.5-4.5 mu M.

5. Use of the test kit according to any one of claims 1 to 4 for the preparation of a kit for predicting the radiation sensitivity of a human.