CN112251505A - TRIP12 gene mutation site detection kit based on Taqman probe method and application thereof - Google Patents

Abstract

The invention relates to a TRIP12 gene mutation site detection kit based on a Taqman probe method, which comprises an amplification primer, a T-type Taqman probe and a C-type Taqman probe; the sequence of the forward primer of the amplification primer is shown as SEQ ID NO.3, and the sequence of the reverse primer of the amplification primer is shown as SEQ ID NO. 4; the C-type Taqman probe is shown as SEQ ID NO.5, and the T-type Taqman probe is shown as SEQ ID NO. 6; 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. The kit for detecting the TRIP12 gene mutation site based on the Taqman probe method can be used for widely and efficiently detecting radiation damage.

Description

TRIP12 gene mutation site detection kit based on Taqman probe method and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a TRIP12 gene mutation site detection kit based on a Taqman probe method 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 radiation injury susceptibility degree of personnel can be accurately distinguished, the post adaptability judgment of the radiation workers is facilitated, and the customization or optimization of the clinical personalized treatment scheme is facilitated.

In addition, the Taqman probe method places the sample and the reaction components in a closed system for reaction, thereby avoiding pollution. The Taqman probe method depends on a PCR method, and has the advantages of high detection speed, high sensitivity and strong specificity. The detection equipment of the method is a fluorescent quantitative PCR instrument which has good popularity and is widely applied to clinical diagnosis and molecular biology experiments, the Taqman probe method does not need any operation after PCR, the operation steps are simple, and the wide application is easy to realize.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a TRIP12 gene mutation site detection kit based on a Taqman probe method, so that radiation damage can be widely and efficiently detected.

In order to achieve the above purposes, the invention adopts the technical scheme that:

a TRIP12 gene mutation site detection kit based on a Taqman probe method comprises an amplification primer, a T-type Taqman probe and a C-type Taqman probe;

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

the C-type Taqman probe is shown as SEQ ID NO.5, and the T-type Taqman probe is shown as SEQ ID NO. 6;

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.

In a preferred embodiment, the invention provides a kit for detecting the TRIP12 gene mutation site based on a Taqman probe method, wherein the TRIP12 gene mutation site is the rs13018957 site of the TRIP12 gene.

In a preferred embodiment, the invention provides a kit for detecting the mutation site of TRIP12 gene based on Taqman probe method, wherein the concentration of the forward primer of the amplification primer is 0.25. mu.M.

In a preferred embodiment, the invention provides a kit for detecting the mutation site of TRIP12 gene based on Taqman probe method, wherein the concentration of the reverse primer of the amplification primer is 0.25. mu.M.

In a preferred embodiment, the invention provides a kit for detecting the mutation site of TRIP12 gene based on Taqman probe method, wherein the concentration of the C-type Taqman probe is 0.2. mu.M.

In a preferred embodiment, the invention provides a kit for detecting TRIP12 gene mutation sites based on a Taqman probe method, wherein the concentration of the T-type Taqman probe is 0.2. mu.M.

The second purpose of the invention is to provide the application of the detection kit in preparing a kit for detecting the susceptibility degree of radiation damage, so that the radiation damage can be widely and efficiently detected.

In order to achieve the purpose, the invention provides the application of the detection kit in preparing the kit for detecting the susceptibility degree of radiation damage.

In a preferred embodiment, the invention provides the use of the above-mentioned detection kit for preparing a kit for detecting susceptibility to radiation injury, wherein the radiation injury is local or systemic adverse reaction of tissues and organs, such as nausea, vomiting, anorexia, leukopenia, skin redness and itching, even carcinogenesis, and the like.

The invention has the beneficial effects that: the TRIP12 gene mutation site detection kit based on the Taqman probe method can be used for detecting radiation damage widely and efficiently.

Drawings

FIG. 1 shows the Taqman probe detection results of the amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 and the T-type SEQ ID NO.5 and the C-type SEQ ID NO.6 in example 2 of the present invention;

FIG. 2 shows the Taqman probe detection results of the amplification primer pair of SEQ ID NO.7 and SEQ ID NO.8 and the T-type SEQ ID NO.9 and the C-type SEQ ID NO.10 in example 2 of the present invention.

Detailed Description

The invention is further described with reference to the following figures and detailed description.

Example 1:

this 1) acquisition and irradiation of sample and analysis of chromosome aberration

Peripheral blood of healthy adult male aged 20-30 years old is collected and irradiated with 0 and 2Gy 60Co 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. By utilizing biological information analysis, the site TRIP12 rs13018957 which is different from the site TRIP12 rs13018957 of the susceptible group and the less susceptible group is screened out, and the table 1 shows.

TABLE 1 selected radiation injury susceptible sites

SNP site	Gene	SNP site position	Alleles
				rs13018957	TRIP12	Chr2:230668858	C/T

Example 2:

1) a probe and primer 2 group is designed aiming at the rs13018957 site of the TRIP12 gene, and is shown in Table 2.

TABLE 2 TRIP12 Gene rs13018957Taqman Probe sequences

2) The method utilizes a blood genome DNA extraction kit (non-centrifugal column type; catalog number: DP319) for 10 human whole blood genome (blood from volunteers, healthy males between 20 and 30 years old) DNA extraction according to the product instructions.

3) And (3) detecting the genotypes of the rs13018957 loci of the TRIP12 genes of 10 samples by using MALDI-TOF mass spectrometry.

4) Using table 2, first set: amplification primer pairs of SEQ ID NO.3 and SEQ ID NO.4, and Taqman probes of T-type SEQ ID NO.5 and C-type SEQ ID NO. 6. And a second group: amplification primer pairs of SEQ ID NO.7 and SEQ ID NO.8, and Taqman probes of T-type SEQ ID NO.9 and C-type SEQ ID NO. 10. Respectively carrying out fluorescent quantitative PCR reaction. A specific reaction system: a10. mu.l reaction system comprising 3.1. mu. l H2O, 5. mu.l TaqMan Fast qPCR Master Mix (2X), 0.25. mu.l Forward Primer (10. mu.M), 0.25. mu.l Reverse Primer (10. mu.M), 0.2. mu.l C-type Taqman probe (10. mu.M), 0.2. mu. l T-type Taqman probe (10. mu.M), 1.0. mu.l template DNA was performed according to the following reaction protocol: 94 ℃ for 3 min; [94 ℃, 5sec, 60 ℃, 30sec ]40 cycles.

5) Comparing the detection result of the MALDI-TOF mass spectrometry with the detection results of two groups of Taqman probe methods, and finding out that the first group: the amplification primer pair of SEQ ID NO.3 and SEQ ID NO.4 and the Taqman probes of T-type SEQ ID NO.5 and C-type SEQ ID NO.6 can accurately detect the C/T mutation at the rs13018957 site of the TRIP12 gene, and the results are shown in Table 3 and FIGS. 1 and 2.

Table 3: comparing the mass spectrum detection result with the detection results of the two groups of probes

The structure of the present invention is not limited to the embodiment described in the specific embodiment, and those skilled in the art can derive other embodiments according to the technical solution of the present invention, and also belong to the technical innovation scope of the present invention.

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Claims (8)

1. A TRIP12 gene mutation site detection kit based on a Taqman probe method is characterized in that the detection kit comprises an amplification primer, a T-type Taqman probe and a C-type Taqman probe;

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

the C-type Taqman probe is shown as SEQ ID NO.5, and the T-type Taqman probe is shown as SEQ ID NO. 6;

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 as claimed in claim 1, wherein the TRIP12 gene mutation site is rs13018957 site of TRIP12 gene.

3. The test kit of claim 1, wherein the forward primer concentration of the amplification primer is 0.25 μ M.

4. The test kit according to claim 1, wherein the concentration of the reverse primer of the amplification primer is 0.25. mu.M.

5. The test kit as claimed in claim 1, wherein the concentration of the type C Taqman probe is 0.2. mu.M.

6. The test kit as claimed in claim 1, wherein the concentration of the T-type Taqman probe is 0.2. mu.M.

7. Use of a test kit according to any one of claims 1 to 6 for the preparation of a kit for testing the susceptibility to radiation damage.

8. Use according to claim 7, characterized in that: the radiation injury is local or systemic tissue organ adverse reaction.

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