crRNA combination for multiple detection of hereditary hearing loss, kit and method theref

Abstract

The invention discloses a crRNA combination for multiple detection of hereditary hearing loss, a kit and a method thereof. The crRNA combination targets five SNP sites of deafness genes GJB2 and SLC26A4 at the same time, and comprises nucleotide sequences as shown in SEQ ID No.2, SEQ ID No.2, SEQ ID No.2, SEQ ID No.2, SEQ ID No.6, SEQ ID No.8 and SEQ ID No.10. According to the multiple detection kit for hereditary hearing loss, a Cas12a detection reaction system is arranged in a micropore of a pore plate, multiple RPA amplification is performed in a hollow honeycomb chip, and the hollow honeycomb chip and the bottom of the micropore are physically isolated by using a hollow gasket, so that two reactions of multiple RPA amplification and Cas12a detection are firstly isolated and then fused; one-step and multiple detection is realized, and aerosol pollution possibly caused by uncovering detection is also avoided. The method also realizes high-throughput detection, at most 96 samples are detected in one reaction, 5 SNP loci are distinguished at the same time, and the method is suitable for large-scale genetic screening.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to the field of SNP detection, in particular to a crRNA combination, kit and method for multiple detection of hereditary deafness, and in particular to a crRNA combination for multiple detection of hereditary deafness based on CRISPR / Cas12a , kits and methods. Background technique [0002] Deafness is a common clinical auditory nervous system defect disease, which can be caused by genetic and environmental factors, of which genetic factors account for about 60% (Morton CC, et al., 2006, N Engl J Med, 354(20): 2151– 2164), there are more than 100 deafness-causing genes discovered by human research ( http: / / hereditaryhearingloss.org / ), but the pathogenesis is mainly concentrated in hotspot mutations in several genes such as GJB2, SLC26A4, 12s rRNA, and GJB3. According to statistics, there are more than 20 million hearing-impaired people in China, 800,000 deaf children ...

AI Technical Summary

Problems solved by technology

Beijing Boao Biotechnology has completed the genetic screening of 2.6991 million newborns across the country by using the deafness gene chip ( http: / / cn.capitalbio.com / gxba / xwzx / gsxw / 2018nyjd / 26885.shtml ), the carrier rate of newborn deafness mutations is about 4-5%, but the disadvantage of this method is that it relies on chip scanners and PCR thermal cyclers, and is limited by the interference between multiple PCR primer pairs, amplification requires Divided into two tubes, the chip hybridization step may cause aerosol contamination, and it is difficult to achieve high-throughput detection, which is not conducive to rapid and efficient large-scale genetic screening

[0005] The existing patent document CN110878343A discloses a Cpf1 (Cas12a) kit and detection method for the rapid detection of the genetic deafness-causing gene SLC26A4 mutation, but its RPA amplification and Cas12a detection are carried out in two steps, and it is easy to open the cover for detection Cause aerosol pollution, and cannot realize simultaneous detection of multiple sites, only one by one, low throughput

In addition, it only targets one gene, SLC26A4, with low coverage

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