CN112251506A - UIMC1 gene mutation site detection kit based on Taqman probe method and application thereof - Google Patents
UIMC1 gene mutation site detection kit based on Taqman probe method and application thereof Download PDFInfo
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Abstract
The invention relates to a UIMC1 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 UIMC1 gene is shown as SEQ ID NO.1, and the sequence of the mutant UIMC1 gene is shown as SEQ ID NO. 2. The UIMC1 gene mutation site detection kit based on the Taqman probe method can be used for widely and efficiently detecting radiation damage.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a UIMC1 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 kit for detecting the mutation site of the UIMC1 gene based on a Taqman probe method, so as to be capable of widely and efficiently detecting the radiation damage.
In order to achieve the above purposes, the invention adopts the technical scheme that:
a UIMC1 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 UIMC1 gene is shown as SEQ ID NO.1, and the sequence of the mutant UIMC1 gene is shown as SEQ ID NO. 2.
In a preferred embodiment, the invention provides a kit for detecting the mutation site of the UIMC1 gene based on a Taqman probe method, wherein the mutation site of the UIMC1 gene is rs1700490 site (mutation from a forward sequence A to G; mutation from a reverse sequence C to T) of the UIMC1 gene.
In a preferred embodiment, the invention provides a UIMC1 gene mutation site detection kit based on a Taqman probe method, wherein the concentration of a forward primer of an amplification primer is 0.25 mu M.
In a preferred embodiment, the present invention provides a UIMC1 gene mutation site detection kit based on the 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 mutation sites of UIMC1 gene based on Taqman probe method, wherein the concentration of the C-type Taqman probe is 0.2 μ M.
In a preferred embodiment, the invention provides a kit for detecting mutation sites of UIMC1 gene based on 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 UIMC1 gene mutation site detection kit based on the Taqman probe method can be used for widely and efficiently detecting radiation damage.
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 UIMC1 rs1700490 locus which is the difference locus between the susceptible group and the non-susceptible group is screened out, and the site is shown in the table 1.
TABLE 1 selected radiation injury susceptible sites
| SNP site | Gene | SNP site position | Alleles |
| rs1700490 | UIMC1 | Chr5:176402401 | G/A |
Example 2:
1) a probe and primer 2 group is designed aiming at rs1700490 site of the UIMC1 gene, and the probe and primer 2 group is shown in a table 2.
TABLE 2 Taqman probe sequence of rs1700490 site of UIMC1 gene
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 rs1700490 sites of 10 samples of UIMC1 genes 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, 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 rs1700490 site of the UIMC1 gene, and the result is shown in Table 3, and figures 1 and 2.
TABLE 3 comparison of the Mass Spectrometry results with the 2 groups of Probe detection results
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.
Sequence listing
<110> China institute for radiation protection
<120> UIMC1 gene mutation site detection kit based on Taqman probe method and application thereof
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Claims (8)
1. A UIMC1 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 UIMC1 gene is shown as SEQ ID NO.1, and the sequence of the mutant UIMC1 gene is shown as SEQ ID NO. 2.
2. The test kit as claimed in claim 1, wherein the mutation site of the UIMC1 gene is rs1700490 site of the UIMC1 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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| CN103103624A (en) * | 2011-11-15 | 2013-05-15 | 深圳华大基因科技有限公司 | Method for establishing high-throughput sequencing library and application thereof |
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