高分辨率熔解曲线法检测药物性耳聋相关线粒体12S rRNA突变

Detection of mitochondrial 12S rRNA mutations related to drug-induced deafness by high-resolution melting analysis

目的建立一种基于高分辨率熔解曲线(HRM)技术的药物性耳聋相关线粒体12S rRNA基因1494C>T和1555A>G突变快速检测方法。方法采用定点诱变克隆策略构建突变质粒DNA标准品;建立突变位点靶序列PCR扩增及HRM分析方法,并检测106例非综合征耳聋患者标本,以DNA直接测序法进行验证。结果建立的HRM检测方法能准确检出线粒体12S rRNA基因1494C>T和1555A>G突变,各基因型HRM曲线特征明显且易于分析判断;106例非综合征耳聋患者标本中检出6例1555A>G突变,检测结果与DNA测序结果一致。结论建立了药物性耳聋相关人类线粒体12S rRNA基因1494C>T和1555A>G突变HRM检测方法,操作简单快速、结果准确可靠,可应用于人群筛查和临床常规分子诊断。

Objective To establish a rapid method for the detection of mitochondrial 12 S rRNA gene 1494 C T and 1555 A G mutations related to drug-induced deafness based on high-resolution melting（ HRM） analysis. Methods Mutational plasmid DNA standard materials were constructed by the site-directed mutagenesis cloning strategy. The PCR-HRM system was developed to detect the mitochondrial 12 S rRNA gene mutations of 106 non-syndromic deafness patients,and the results were further verified with DNA sequencing. Results The established method could detect the mitochondrial 12 S rRNA gene 1494 C T and 1555 A G mutations correctly,and the HRM curves of each genotype were obvious and easy to be analyzed. Among 106 patients with non-syndromic deafness,6 were detected A1555 G mutation,and the results were consistent with those of DNA sequencing. Conclusion The HRM method for the detection of mitochondrial 12 S rRNA gene 1494 C T and 1555 A G mutations related to drug-induced deafness is successfully established,which is simple,rapid and accurate,and may be applied to the mutation screening and clinical molecular diagnosis.