碱基淬灭探针技术单管检测线粒体DNA 12Sr RNA A1555G及C1494T突变

A novel method of detecting mitochondrial C1494T and A1555G mutations by using the base- quenched probe technique in a single PCR assay

目的应用碱基淬灭探针技术建立单管同时检测线粒体DNA12Sr RNA A1555G和C1494T突变的方法。方法探针的一端标记6-羧基荧光素，同时构建4个载体做为扩增模板，分别代表可能的4种基因型组合。对117例耳聋患者外周血标本进行线粒体DNA12Sr RNA A1555G和C1494T突变的检测，同时选取15例样本进行DNA测序，验证所建立方法的可靠性和准确性。结果根据熔解曲线可以清晰地对线粒体DNA12Sr RNA1555位点和1494位点的基因型做出判断。117例耳聋患者中3例携带A1555G突变（2．56％），1494位点未检出突变；基于碱基淬灭探针技术的单管检测方法与DNA测序的结果一致。结论碱基淬灭探针单管检测技术可用于临床线粒体DNA突变耳聋基因检测。

Objective In the present study, we described a new method to detect the mitochondrial DNA （mtDNA） A1555G and C1494T mutations by using the base-quenched probe technique in a single PCR reaction. Methods 6-carboxyfluoresceint （FAM） was directly conjugated to the 3＇ end of the probe. Four vectors, representing the four possible genotype combinations, were constructed as the amplification templates for the methodology established. In the present study A1555G and C1494T mutations in 117 individuals with hearing loss were detected by the base-quenched probe method and were further validated by the direct DNA sequencing analysis. Results From the melting curve, we could distinguish the four haplotypes accurately. And there were complete concordance between the base-quenched probe method and direct DNA sequencing. Conclusion This method is suitable for clinical test of mtDNA A1555G and C1494T mutations in individuals with hearing loss.