Method for detecting GJB2 gene mutation by fluorescence quantitative PCR technology, and kit thereof

Abstract

The invention provides a method for detecting 235delC mutation of deafness related gene GJB2 by a fluorescence quantitative PCR technology, and a kit thereof. According to the kit, specific oligonucleotide primers are designed according to the 235delC mutational site of the GJB2 gene; a fluorescent substance is adopted, and a sample requiring detection is detected respectively by a wild type fluorescent PCR system and a mutant type fluorescent PCR system; the Ct values of the two reactions and the different value (DeltaCt) of the two Ct values are compared to judge whether the 235delC mutation of the GJB2 gene exists. With adopting the method and the kit of the present invention, the valuable clinical reference information can be provided for the etiological diagnosis of the deafness patient, and the pregnant couple with the possibility of deafness carriers can be screened so as to provide assistance for prenatal diagnosis of the deafness. The kit of the present invention adopts the special PCR reaction system and the special reaction conditions, and specifically detects the 235delC mutational site of the GJB2 gene, such that the kit has advantages of high throughput and automatic tube closing operation, and is applicable for the clinical laboratories.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and particularly relates to a method for detecting the mutation of deafness-related gene GJB2 235delC by primer-specific real-time quantitative PCR technology and a kit thereof. Background technique [0002] The incidence of deafness is high. It is currently confirmed that about 60% of deafness is caused by genetic factors. Deafness caused by genetic factors, 30% of patients belong to syndromic deafness (SHI), and 70% of patients belong to non-syndromic deafness (NSHI) . Autosomal recessive inheritance accounts for 75%-80% of NSHI, that is, both parents of the child have normal hearing, but they are both positive carriers of the disease-causing gene mutation, mostly sporadic. Currently, it is believed that about 50% of autosomal recessive NSHI is inherited It is caused by GJB2 gene mutation, and there are different hotspot pathogenic sites in people of different races and regions. For Asia...

AI Technical Summary

Problems solved by technology

Direct sequencing of PCR products is currently the most widely used and more accurate method, but this method requires expensive instruments and professional personnel to operate, time-consuming and laborious, and it is difficult to promote in clinical practice

The gene chip method has the advantages of fast and high throughput, but the operation is complicated, there are many steps, the cost is high and it is not easy to popularize

The method of detecting gene mutation with restriction fragment length polymorphism (RFLP) has the advantage of low cost, but the process is cumbersome and takes a long time and is not suitable for popularization

The published patent (Patent No. 200810222745.7) adopts allele-specific PCR technology and uses 4 primers in a PCR reaction system, which has advantages for multi-site detection, but this method requires subsequent gel after the PCR reaction is completed. The results of electrophoresis analysis have disadvantages such as open operation, gel electrophoresis pollutes the environment, etc., and are not suitable for clinical promotion

Images