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Kit for detecting mutation of deafness susceptibility genes and application of kit

A technology of susceptibility genes and kits, which is applied in the field of kits for detecting mutations of deaf susceptibility genes, can solve the problems of high cost, long time-consuming detection of deaf susceptibility genes, and low accuracy, and achieve low cost and saving reaction materials , time-consuming effect

Active Publication Date: 2017-09-19
GUANGZHOU HYBRIBIO MEDICINE TECH LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, to provide a deafness susceptibility gene detection kit based on multiple asymmetric PCR and melting curve analysis technology and its application, aiming to solve the problem that the existing deafness susceptibility gene detection takes a long time. , low accuracy and high cost technical problems

Method used

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  • Kit for detecting mutation of deafness susceptibility genes and application of kit
  • Kit for detecting mutation of deafness susceptibility genes and application of kit
  • Kit for detecting mutation of deafness susceptibility genes and application of kit

Examples

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Effect test

Embodiment 1

[0047] A kit for detecting deafness susceptibility gene mutations. The primers and probes in the kit are designed according to the human GJB2, SLC26A4, mitochondrial 12SrRNA, GJB3 and RPP30 gene sequences in Genebank.

[0048] Among them, the design method of amplification primers is to use Primerpremier 5 software to design amplification primers for each site after downloading the corresponding sequences from Genebank. Add a universal primer sequence at the 5' end of the reverse primer (select a sequence of about 20 bp, which has a low similarity to the human gene sequence and cannot produce amplified fragments for the human genome after testing), and add a balanced sequence at the 5' end of the forward primer sequence (The length is close to that of the same primers, the sequence similarity to the human gene sequence is low, and the sequence that cannot generate amplified fragments for the human genome is detected). Follow the general principles of hybridization probe design...

Embodiment 2

[0051] A test kit for detecting deafness susceptibility gene mutations, the kit includes PCR reaction solution (pre-packed with fluorescent PCR eight-tube tubes, each tube contains 35 μL of PCR reaction solution, including the primers and probes in Example 1), EL positive control 1 (equal mixture of 9 wild-type sequences and internal reference genes), EL positive control 2 (equal mixture of 235delC mutant sequence, 7 wild-type sequences and internal reference genes) , EL positive control 3 (equal mixture of 1555A>G site mutant sequence, 7 site wild-type sequences and internal reference genes), EL negative control (Tris-EDTA buffer). Wherein, the PCR reaction solution includes: 1×PCR buffer (including Tris-HCl, KCl and a volume ratio of 50% glycerol), dNTP0.25mM, MgCl 2 1.5mM, EL enzyme 0.2U / μL, the concentration of the first eight pairs of primers is 0.1μM, the concentration of the ninth primer is 2μM, and the concentration of 11 probes is 0.25μM.

[0052] The fluorescent PCR...

Embodiment 3

[0058] Carry out detection and analysis with the kit of embodiment 2, the instrument used is SLAN 96S real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.), concrete steps are as follows:

[0059] (1) Utilize the nucleic acid extraction kit (Shenzhen United Medical Technology Co., Ltd., record number: Yueshen Machinery No. 20160073) to extract the genomic DNA of 4 people's whole blood samples (EDTA anticoagulated blood); German Implen NanoPhotometer TM P-Clas micro spectrophotometer) to measure the OD of DNA 260 、OD 280 and concentration: OD 260 / OD 280 The value is between 1.5 and 2.0, and the DNA concentration ranges from 10ng / μL to 100ng / μL; the DNA samples thus obtained can be used for mutation detection.

[0060] (2) Take an eight-tube tube of pre-packed PCR reaction solution from the kit, add 5 μL of the DNA extracted in step (1) to the first four tubes in turn, and add the EL-positive DNA in the kit to the other four tubes. Control 1,...

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Abstract

The invention belongs to the technical field of gene detection and particularly relates to a kit for detecting mutation of deafness susceptibility genes and an application of the kit. A PCR mixture in the kit contains 17 primer single strands shown as SEQ ID NO: 1-17 and 11 probes shown as SEQ ID NO: 19-29, genotypes of 9 mutation sites of four deafness susceptibility genes and one reference gene can be detected simultaneously in a single-tube PCR system, the genotype of a sample is acquired through fluorescent PCR melting curve analysis after PCR amplification is ended, the whole operation can be finished within 2-3 h, a few operation steps are adopted, short time is consumed, many single-tube detection sites are adopted, and the flux and mutation coverage are high; besides, the kit adopts homogeneous-phase detection and closed-tube operation, so that pollution of a PCR product is reduced, detection specificity is high, and a result is easy to interpret.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a kit for detecting deafness susceptibility gene mutation and its application. Background technique [0002] Deafness is a common disease that seriously affects the quality of human life. Worldwide, there is an average of 1 child with congenital deafness in every 1,000 newborns. According to the results of the second national sample survey of disabled people at the end of 2006, there were 26.7 million people with hearing disabilities in China, accounting for 19.3% of the total number of disabled people; there were 800,000 hearing-impaired children aged 1-7, and more than 30,000 deaf children were born every year. Deafness can be caused by environmental factors (such as medical factors, environmental exposure, trauma, drugs, etc.), or by mutations in related genes or the combined effects of genes and the environment, of which genetic factors account for 60%. Sc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2531/107C12Q2537/143C12Q2527/107C12Q2563/107C12Q2545/101
Inventor 谭爱女郭永超王艳平周代志雷湘华
Owner GUANGZHOU HYBRIBIO MEDICINE TECH LTD
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