Mutation Detection Using Real-Time Quantitative Polymerase Chain Reaction With Molecular Beacon For Chinese Inherited Deafness Screening

Aim: This study was carried out to establish a new method which combinesmolecular beacon and Real-time PCR to screen deafness carrier status of32mutationswhich frequently found in Chinese inherited deafness groups. This new method is muchcheaper, faster and easier. One hand, this new method could screen the32mutations forinherited deafness patients, on the other hand, this new method could prevent inheriteddeafness to happen. It has an important meaning to take this detection before marriage andantepartum.Method: First we found the mutations which frequently found in Chinese inheriteddeafness groups from papers. Downloaded the DNA sequences from NCBI, and then wedesigned the primers against them. The DNA sequences were clone into PMD18vector,then we got the mutation vectors use site-specific mutagenesis. After that we screen theallele-specific primers using wild-type and mutant-type vectors. Then we designeddetection probe attached with FAM and control probe attached with ROX. Whether thesample is a mutation carrier was inferred by the control-normalized threshold difference(△△Ct). We screened73DNA samples from inherited deaf patients with this method. Theresults were perfectly marched with that genotyping with direct sequencing.Conclusion: The results indicate that this simple, rapid, reliable molecular beacon based-RT-PCR with low cost is suitable for inherited deaf gene mutation carrier screening.