Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples

作者全名：Tan, Jiefeng; Zhang, Xiaoqing; Wei, Xue; Ding, Min

作者地址：[Tan, Jiefeng; Zhang, Xiaoqing; Wei, Xue; Ding, Min] Chongqing Med Univ, Sch Lab Med, Key Lab Clin Lab Diagnost, Minist Educ China, Chongqing 400016, Peoples R China

通信作者：Ding, M (通讯作者)，Chongqing Med Univ, Sch Lab Med, Key Lab Clin Lab Diagnost, Minist Educ China, Chongqing 400016, Peoples R China.

来源：CURRENT ISSUES IN MOLECULAR BIOLOGY

ESI学科分类：MOLECULAR BIOLOGY & GENETICS

WOS号：WOS:001255054800001

JCR分区：Q3

影响因子：2.8

年份：2024

卷号：46

期号：6

开始页：5454

结束页：5466

文献类型：Article

关键词：amplification refractory mutation system; nucleic acid detection; genotyping; deafness; single nucleotide polymorphism

摘要：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/mu L. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/mu L. 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.

基金机构：Natural Science Foundation Project of Chongqing [cstc2020jcyj-msxm-X0141]

基金资助正文：This research was funded by the Natural Science Foundation Project of Chongqing (Grant Number cstc2020jcyj-msxm-X0141)