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Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof

A thyroid stimulating hormone and kit technology, which is applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of long time, 2-3 days, expensive testing equipment, and not suitable for clinical use.

Active Publication Date: 2013-08-07
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there are mainly two levels for the detection of mutation sites, one is the indirect detection at the protein level; the other is the direct detection of genetic material, including SSCP, RFLP, HTX, DGGE, DC, DHPLC, sequencing, etc. These methods have their own advantages and disadvantages, some require expensive detection equipment, and some have a long operation cycle, requiring 2-3 days to produce results, which are not suitable for clinical TSHR mutation detection

Method used

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  • Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof
  • Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof
  • Kit for detecting gene mutational sites of TSHR (thyroid stimulating hormone receptor), and use method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0058] The preparation of embodiment 1 positive control substance

[0059] Based on the TSHR gene sequence with NCBI registration number: NM_000369 (GI: 64085120), an upstream primer TSHR-F (SEQ ID NO: 1) and a downstream primer TSHR-R (SEQ ID NO: 2) were designed. These two primers were used to amplify the TSHR gene of normal people, and the gene sequence is NCBI registration number: NM_000369 (GI: 64085120). After purification, the obtained PCR product was cloned into the pMD18-T vector by TA and identified by sequencing, and the plasmid verified by sequencing was transformed into DH5α clone to obtain a large number of recombinant plasmids; the recombinant plasmid was diluted to serve as the wild-type positive control substance. The sequence amplified by the primers in the wild-type positive control product is SEQ ID NO:9.

[0060] Using the recombinant plasmid as a template, the multi-site-directed mutagenesis kit of Stratagene was used to perform site-directed mutation on...

Embodiment 2

[0061] Embodiment 2 kit detects

[0062] Through the thyroid-stimulating hormone receptor gene mutation site detection kit and the use method of the kit of the present invention, the samples of 5 experimenters were tested for the thyroid-stimulating hormone receptor (TSHR) gene mutation site, and the specific steps were as follows:

[0063] 1. Preparation of DNA samples to be tested: Genomic DNA was extracted from 5 plasma samples using the Genome Extraction Kit;

[0064] 2. Prepare reagents: take 36 μl TSHR 623 site nucleic acid fluorescence PCR mixture and 0.4 μl Taq enzyme in each tube, shake and mix, and centrifuge at 3000 rpm for a few seconds to obtain a blend of TSHR 623 site nucleic acid fluorescence PCR mixture and Taq enzyme; Take 36 μl TSHR 631 site nucleic acid fluorescence PCR mixture and 0.4 μl Taq enzyme, shake and mix, and centrifuge at 3000rpm for a few seconds to obtain a blend of TSHR 631 site nucleic acid fluorescence PCR mixture and Taq enzyme;

[0065] 3...

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Abstract

The invention discloses a kit for detecting gene mutational sites of a TSHR (thyroid stimulating hormone receptor), and a use method and application thereof. The kit comprises 1) fluorescence PCR detection mixed liquid, 2) Taq enzyme, 3) a TSHR wild type positive comparison product, 4) a TSHR mutation type positive comparison product, and 5) ddH2O. The kit detects coding genes of 623 sites and / or631 sites of TSHR proteins by adopting a fluorescence quantitative PCR method, and judges whether the coding genes are mutated or not so as to judge whether the 623 sites and / or 631 sites of the TSHR proteins are mutated or not. The mutation of the protein sites can be used for aided diagnosis and prevention of thyroid diseases. The kit has the characteristics of real-time detection, quantitative and high-throughput detection and the like, is easy to operate, and has high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of gene mutation detection, in particular to a detection kit for the mutation site of the thyroid-stimulating hormone receptor gene, a use method and an application. Background technique [0002] The main physiological functions of the thyroid gland are controlled by the action of thyroid stimulating hormone (TSH) on the thyroid stimulating hormone receptor (TSHR) on the thyroid cell membrane. TSHR is a member of the G protein-coupled receptor family, and the amino acid terminal of the extramembrane region It is very long and is the main part that determines the specific binding with TSH and TSAB. The main function of TSHR is to combine with TSH, cause a series of biological reactions through the adenylate cyclase (AC)-CAMP pathway, promote the secretion of thyroid hormones, improve the iodine uptake capacity of the thyroid gland, and promote the synthesis and release of thyroid hormones. It plays a very important ro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 丁钢强楼晓明朱文明朱旭平朱勤玮邵俊斌
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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