实时荧光定量Taqman探针法检测线粒体基因1555A〉G突变  

Real-time Taqman probe technique system for detecting the MtDNA 1555 A 〉 G mutation

作　　者：袁永一[1] 黄德亮[1] 韩东一[1] 金政策 戴朴[1] 

机构地区：[1]解放军总医院耳鼻咽喉头颈外科解放军总医院耳鼻咽喉科研究所聋病分子诊断中心,北京100853 [2]山东三月三(澳麦尔)基因技术有限公司

出　　处：《中华耳鼻咽喉头颈外科杂志》2009年第2期160-164,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：基金项目：国家自然科学基金（30572015）;北京市自然科学基金（7062062）

摘　　要：目的建立以实时荧光定量Taqman探针技术检测线粒体DNA1555A〉G突变的方法，实现更快速、简便、准确筛查这一突变的目标。方法设计针对线粒体DNA1555A〉G突变的Taqman探针和引物，摸索建立稳定的检测方法，同时进行可靠性验证。从解放军总医院聋病分子诊断中心耳聋DNA库选取标本132例，遵循双盲法原则，分别以实时荧光定量Taqman普通探针法、试剂盒法和直接测序法检测线粒体DNA1555A〉G突变，将三种方法检测的结果进行比对。结果132例耳聋病例经实时荧光定量Taqman普通探针法检测发现线粒体DNA1555A〉G突变阳性患者32例，阴性100例，与试剂盒法和直接测序法检测结果完全相符，未发现假阳性和假阴性。结论实时荧光定量Taqman探针技术检测线粒体DNA1555A〉G突变的方法具有检测结果准确直观、简单省时（反应时间由原来的最快6h缩短到1．5h），特异性强，敏感性高的优点，适用于对母系遗传性耳聋线粒体DNA1555A〉G突变的大规模筛查或氨基糖甙类抗生素应用前预防性检测。Objective To establish a Real-time Taqman probe technique system to detect the mtDNA 1555A 〉 G mutation in deaf population. Methods Primers and Taqman probes for mtDNA 1555A 〉 G mutation were designed and synthesized. The technique system for detecting mtDNA 1555A 〉 G mutation using Real-time Taqman probes was established. Then the reliability of the technique was tested in 132 patients with severe to profound bearing loss who were detected for the mtDNA 1555A 〉 G mutation by sequencing, Kit method and Real-time Taqman probe technique at the same time. Finally, the results by the above three ways were compared. Results Thirty-two cases with mtDNA 1555A 〉 G mutation were found by the technique of Real-time Taqman probe. These findings coincided with the results from sequencing and Kit method completely. Both the false positive rate and the false negative rate were zero. Conclusions The technique possesses the merits of accuracy, conveniency, high sensitivity, high specificity and intuitionistie results, etc. Importantly, the Real-time Taqman probe technique only needs 1.5 hours to detect the 1555A 〉 G mutation and it saves 4. 5 hours for one reaction compared with the Kit method popularly used nowadays. The technique system of detecting mtDNA 1555A 〉 G mutation is reliable. It＇s suitable for large- scale detecting and preventive diagnosis of mtDNA 1555A 〉 G mutation.

关 键 词：听觉丧失 DNA 线粒体 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]