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目的利用Taq Man探针建立熔解曲线定量分析技术平台,比较Taq Man探针熔解曲线技术、变性高效液相色谱技术(DHPLC)及测序技术在检测线粒体DNA 1555A>G异质性突变负荷方面的差异。方法建立Taq Man探针熔解曲线技术,分析氨基糖苷类药物性耳聋家系及正常人群的线粒体DNA 1555A>G异质性突变水平,并与前期实验中DHPLC技术及测序技术的检测结果进行对比分析。结果 Taq Man探针熔解曲线技术成功分析50例样本的线粒体DNA 1555A>G突变水平,相比于Sanger测序技术多发现1例异质性突变,但比DHPLC技术少发现3例异质性突变。结论本实验建立的Taq Man探针熔解曲线技术成功检出19%至86%范围内的线粒体DNA 1555A>G异质性突变水平,操作简单,成本低。
OBJECTIVE To establish a melting curve quantitative analysis platform using Taq Man probe and compare the TaqMan probe melting curve technique, denaturing high performance liquid chromatography (DHPLC) and sequencing techniques in detecting mitochondrial DNA 1555A> G heterogeneous mutation load . Methods Taq Man probe melting curve technique was established to analyze the mitochondrial DNA 1555A> G heterozygous mutation level in the aminoglycoside drug-induced deafness families and normal population. The results were compared with the results of DHPLC and sequencing. Results TaqMan probe melting curve technique successfully analyzed mitochondrial DNA 1555A> G mutation level in 50 samples. Compared with Sanger sequencing technique, one more heterogeneous mutation was found, but three heterozygous mutations were found less than DHPLC technique. Conclusion The TaqMan probe melting curve technique established in this study successfully detected mitochondrial DNA 1555A> G heterozygous mutation within the range of 19% to 86%, which was simple in operation and low in cost.