Multiple PCR specific primers, kit and method for detecting hereditary hearing loss gene based on high throughput sequencing technique, kit and method

Abstract

The invention belongs to the field of gene detection and especially relates to multiple PCR specific primers, kit and method for detecting a hereditary hearing loss gene, based on a high throughput sequencing technique. The primers include 52 pairs of specific primers, the nucleotide sequences of which are shown as SEQ ID NO:1 to SEQ ID NO:104. The kit further comprises the specific primers, a universal primer, an enzyme mixture, a quality control substance and nuclease-less water. The invention also discloses a detection method for capturing and amplifying the hereditary hearing loss gene through once PCR amplified reaction by utilizing the primers or kit. According to the scheme provided by the invention, the detection efficiency is effectively increased, the accuracy is increased, the cost is lowered and the operation step is simplified.

Description

technical field [0001] The invention belongs to the field of gene detection, in particular to a multiple PCR specific primer, a kit and a detection method for detecting hereditary deafness genes based on high-throughput sequencing technology. Background technique [0002] Deafness is a common clinical birth defect. In China, there are more than 27 million patients with hearing and speech disabilities, ranking first among all kinds of disabilities. Among newborns, the incidence of congenital hearing impairment is as high as 1-3 / 1000. It is estimated that 30,000 to 60,000 new deaf patients are added every year in my country, and about 60% of these deafness disabilities are caused by genetic factors. [0003] Genetic detection of hereditary deafness is to guide the intervention and treatment of deaf patients or high-risk groups by detecting the deafness-causing genes of the subjects. Risk, gene carrying risk, disease risk of offspring, etc. to make scientific assessments to gu...

AI Technical Summary

Problems solved by technology

Sanger sequencing can only sequence a certain region of a sample at a time, which has low detection efficiency, high cost, and low sensitivity (20%)

Gene chip method and fluorescent quantitative PCR method are used to detect deafness genetic disease genes. This method can only design detection probes for one or several known mutation sites to form a detection chip, so it cannot be used to detect unknown mutation sites. It is not comprehensive and accurate enough

Multi-color fluorescent melting curve analysis method, the fluorescent dye used is a general-purpose double-stranded DNA intercalator, which cannot recognize primer dimers and non-specific amplification products, and may easily cause false positives

The high-throughput sequencing method is mainly provided by the sequencing instrument provider for the sequencer supporting library construction kit, which has passed the strict quality control evaluation of the supplier company and has a good library construction effect, but the disadvantage is that the delivery time of the kit is long and the reagents Box packaging specifications are fixed, testing flexibility is not enough, etc.

Some reagent manufacturers build libraries according to the experimental methods shared and reported in the literature, but the results of the completed experiments are uneven, and the quality of the sequencing is not high, which affects the clinical reference value

[0005] At present, the mainstream detection of deafness genes on the market is GJB2 (gene mutation clinically manifested as congenital severe deafness), GJB3 (gene mutation clinically manifested as acquired high-frequency sensorineural deafness), SLC26A4 (gene mutation clinically manifested as congenital or delayed Onset deafness), MT-RNR1 (gene mutation is clinically manifested as drug-induced deafness, and individuals carrying gene mutations are sensitive to aminoglycoside drugs, which is often said to be "one-shot deafness") 4 common hereditary deafness genes, and detection Most of the sites are screened, and the detected gene mutation information is insufficient, which is not enough to reflect the overall situation of genetic deafness mutations

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