Abstract：
A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.
摘要：
线粒体DNA（mtDNA）1555A>G中的单核苷酸变体与药物诱导的听力损失有关。对于1555A>G突变位点，构建了1555A野生型和1555G突变型质粒,分别。在这项研究中,提出了一种基于TaqMan扩增难治性突变系统的PCR方法来检测mtDNA1555A>G。常见的上游引物，一个普通的TaqMan探测器,并设计了两个具有错配碱基的下游等位基因特异性引物。通过两个反应实现了耳聋相关基因1555位点野生型和突变型的一步扩增和检测。基于这种检测方法,野生型和突变型质粒检测系统的最低检测限为50拷贝/μL.在真实干血斑(DBS)样品中检测核酸的最小灵敏度为0.1ng/μL。在正常的DBSDNA样本中,突变丰度的检测限达到0.78%。检测方法的特异性为100%,变异系数小于3.36%。使用从113例新生儿DBS样品中提取的临床DNA验证了该方法。此外,它显示与双向Sanger测序100%一致。可作为临床检测耳聋相关基因的一种可选方法。
