HRM method and kit for clinically detecting deafness-related gene mutation

Abstract

The invention discloses an HRM method and kit for clinically detecting deafness-related gene mutation. The HRM method comprises the steps that PCR amplification and HRM scan analysis are conducted by extracting DNA of a sample to be detected and applying primers shown in SEQ ID NO.1-30; the deafness-related gene mutation type is judged according to a scan analysis result, a sample free of mutation peaks in HRM scan typing is negative, and a sample with the mutation peaks in HRM scan typing is positive. The kit comprises the primers, quality control products, a PCR reagent, fluorescent dyes and the like; the kit has the advantages that all the primers in the kit can conduct a PCR reaction at the same temperature, the detection sensitivity and the specificity are high, a small reaction system is achieved, the detection sample source is rich, reacting is easy, convenient and rapid, the reagent cost is low, and high throughput is achieved. The HRM method and kit are suitable for clinical neonatal hereditary deafness gene screening and pre-pregnancy deafness gene screening for good prenatal and postnatal care and beneficial for guiding good prenatal and postnatal care and clinical personalized medication.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an HRM method and a kit for clinical detection of five common deafness-related gene mutations. Background technique [0002] Deafness is a global human sensory nerve defect, affecting the health of nearly 600 million people. About 2 to 6 of every 1,000 children suffer from severe hearing loss at birth or in childhood. Genetic factors play an important role in the pathogenesis . About 80% of hereditary deafness is non-syndromic hearing loss (NSHL), that is, deafness without other symptoms, of which autosomal recessive non-syndromic hearing loss (ARNSHL) accounts for 80% of NSHL . So far, as many as 46 mutation sites of deafness genes related to ARNSHL have been reported (Hilgert N, Smith RJ, Van CampG. Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics, Mutation research 2009; 681:189-196.), the most common dea...

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Problems solved by technology

[0006] At present, there are two commonly used clinical methods for the detection of GJB2, SLC26A4, and mtDNA12SrRNA gene mutation sites: one is direct DNA sequencing. The operation is cumbersome, and the sensitivity is not high (only mutations with a mutation frequency higher than 25% can be detected); the second is the gene chip method (Boao Biological Co., Ltd.), which is characterized by high throughput, but there is a positive detection rate and accurate The rate is lower than the traditional method (especially the SLC26A4 gene mutation site), the reagent consumption is large (25μL reaction system), the detection time is long, and the cost is high. Clinical application research of mutation microarray diagnostic chip. Chinese Journal of Otology, 2014:6-10)

In addition, there are fluorescence quantitative methods, restriction fragment length polymorphism analysis, denaturing high-performance liquid chromatography, etc., but these methods have shortcomings such as long detection time, cumbersome operation, and low sensitivity.

With the introduction of the national two-child policy and the increasing popularity of newborn birth defect screening, the existing deafness-related gene mutation detection methods can no longer meet clinical needs, and there is an urgent need to develop a fast, accurate, low-cost, high-sensitivity deafness Related gene detection technology

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