CN111154859A - TRIP12 gene mutation site detection kit and application thereof - Google Patents
TRIP12 gene mutation site detection kit and application thereof Download PDFInfo
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- CN111154859A CN111154859A CN202010051752.6A CN202010051752A CN111154859A CN 111154859 A CN111154859 A CN 111154859A CN 202010051752 A CN202010051752 A CN 202010051752A CN 111154859 A CN111154859 A CN 111154859A
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention belongs to the technical field of biological detection, and relates to a TRIP12 gene mutation site detection kit 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 TRIP12 gene is shown as SEQ ID NO.1, and the sequence of mutated TRIP12 gene is shown as SEQ ID NO. 2. The TRIP12 gene mutation site detection kit can detect radiation damage with high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of biological detection, and relates to a TRIP12 gene mutation site detection kit and application thereof.
Background
At present, about 20 thousands of radiation workers exist in China. Radiation workers are required to receive occupational irradiation for a long time during daily work, and the injury of accumulated irradiation to susceptible people is obvious, so that an effective method is needed for judging whether the personnel meet the post requirements or not so as to reduce occupational injury.
In addition, radiation therapy is one of the main therapeutic approaches to malignancy, and over 50% of cancer patients require radiation therapy. However, even with the same radiation treatment regimen, the degree of radiation damage varies from patient to patient, and the cause of this is mainly related to the individual differences in susceptibility to radiation damage: under the same treatment condition, people susceptible to radiation damage may cause serious adverse reactions, and people not susceptible to radiation damage may have a risk of poor treatment effect.
Therefore, the method can accurately distinguish the person susceptible to radiation damage, is favorable for the post adaptability judgment of the radiation worker, and is also favorable for customizing a clinical personalized treatment scheme or optimizing the treatment scheme.
The TRIP12rs13018957 locus is located on chromosome 2, the gene of the locus is a thyroid hormone receptor interaction factor, and the protein encoded by the gene is E3 ubiquitin protein ligase and participates in the degradation of p19ARF/ARF subtype of tumor suppressor CDKN 2A. The encoded protein also plays a role in DNA damage response by modulating the stability of USP 7.
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 susceptibility of radiation damage, so that susceptible people can be quickly and accurately distinguished with high flux and low cost.
Disclosure of Invention
The invention aims to provide a kit for detecting TRIP12 gene mutation sites, which can detect radiation damage with high efficiency and low cost.
To achieve the object, in a basic embodiment, the present invention provides a kit for detecting a mutation site of TRIP12 gene, the kit comprising 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 TRIP12 gene is shown as SEQ ID NO.1, the mutation site is rs13018957 site of TRIP12 gene (mutation from C to T), and the sequence of the mutated TRIP12 gene is shown as SEQ ID NO. 2.
In a preferred embodiment, the invention provides a kit for detecting the mutation site of TRIP12 gene, wherein the concentration of the forward primer of the amplification primer is 2.0-3.0. mu.M.
In a preferred embodiment, the present invention provides a kit for detecting a mutation site of TRIP12 gene, wherein the concentration of the reverse primer of the amplification primer is 2.0-3.0. mu.M.
In a preferred embodiment, the present invention provides a kit for detecting a mutation site of TRIP12 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 detecting radiation damage, so that the radiation damage can be detected 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 the detection of radiation damage.
In a preferred embodiment, the present invention provides the use of the above-mentioned detection kit for the preparation of a kit for detecting radiation injury, wherein the radiation injury is a local or systemic adverse reaction of a tissue or organ (e.g., nausea, vomiting, anorexia, leukopenia and/or red and itchy skin, etc.).
The kit has the beneficial effects that the kit for detecting the TRIP12 gene mutation site can detect radiation damage with high efficiency and low cost.
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.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/human_ g1k_v37.fasta.gz. By utilizing biological information analysis, the site TRIP12rs13018957 which is the difference site between the susceptible group and the less susceptible group is screened out, and the table 1 shows.
TABLE 1 selected radiation injury susceptible sites
Example 2:
1) the method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) human whole blood genome (derived from step 1) of example 1) 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 SAP Buffer (10X), 0.3. mu.l SAP enzyme (1U/. mu.L)) were applied to the reaction product of step 2) according to the following procedure: at 37 ℃ for 40 min; 85 ℃ for 5 min. 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 (10X), 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) The extension product from step 4) was purified (6 mg of resin was uniformly covered on 384 well plates 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, centrifuged at 3500rpm for 5 minutes, and then prepared).
6) Detecting the genotype of the gene locus: transferring the sample treated in step 5) to MassARRAYPectroCHIP chip (MassArray)TMNanodispenser, SAMSUNG), and put into a mass spectrometer (massarrycompact System, SEQUENOM) for detection.
The detection result shows that the primer designed by the embodiment can detect the genotype of the locus rs13018957 of the TRIP12 gene and can be used for detecting the mutation of the locus TRIP12rs13018957 of the radiation injury susceptibility gene.
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> TRIP12 gene mutation site detection kit and application thereof
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Claims (6)
1. A kit for detecting TRIP12 gene mutation sites 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 TRIP12 gene is shown as SEQ ID NO.1, and the sequence of the mutated TRIP12 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 2.0-3.0 mu M.
3. The detection kit according to claim 1, characterized in that: the concentration of the reverse primer of the amplification primer is 2.0-3.0 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 the detection of radiation damage.
6. Use according to claim 5, characterized in that: the radiation injury is local or systemic tissue organ adverse reaction.
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| CN202010051752.6A CN111154859A (en) | 2020-01-17 | 2020-01-17 | TRIP12 gene mutation site detection kit and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010051752.6A CN111154859A (en) | 2020-01-17 | 2020-01-17 | TRIP12 gene mutation site detection kit and application thereof |
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| CN111154859A true CN111154859A (en) | 2020-05-15 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101258250A (en) * | 2005-07-07 | 2008-09-03 | 拜奥默里克斯股份有限公司 | Method for breast cancer diagnosis |
| US20130225433A1 (en) * | 2012-02-29 | 2013-08-29 | The Regents Of The Universitys Of Michagan | Prostate cancer markers and uses thereof |
| CN109402132A (en) * | 2018-11-29 | 2019-03-01 | 福州福瑞医学检验实验室有限公司 | It is a kind of encode SCN1A gene mutation body nucleic acid and its application |
-
2020
- 2020-01-17 CN CN202010051752.6A patent/CN111154859A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101258250A (en) * | 2005-07-07 | 2008-09-03 | 拜奥默里克斯股份有限公司 | Method for breast cancer diagnosis |
| US20130225433A1 (en) * | 2012-02-29 | 2013-08-29 | The Regents Of The Universitys Of Michagan | Prostate cancer markers and uses thereof |
| CN109402132A (en) * | 2018-11-29 | 2019-03-01 | 福州福瑞医学检验实验室有限公司 | It is a kind of encode SCN1A gene mutation body nucleic acid and its application |
Non-Patent Citations (5)
| Title |
|---|
| EMBL-EBI: "rs13018957", 《ENSEMBL RELEASE 98》 * |
| JUANCONG DONG: "Single nucleotide polymorphisms of radiation susceptible genes and radiosensitive miRNA and LncRNA in Chinese population", 《ARADOS MEETING》 * |
| L WANG等: "TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects", 《ONCOGENE》 * |
| 李建国等: "不同剂量γ射线照射人肝细胞差异基因表达谱的研究", 《辐射防护》 * |
| 王放等: "功能基因组学及其在辐射生物效应研究中的应用", 《辐射防护通讯》 * |
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