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实时荧光定量Taqman探针法检测线粒体DNA1494C〉T突变技术的建立及应用研究

ISSN号：1006-7299
期刊名称：《听力学及言语疾病杂志》
时间：0
分类：R764.43[医药卫生—耳鼻咽喉科;医药卫生—临床医学]
作者机构：[1]中国人民解放军总医院耳鼻咽喉-头颈外科中国人民解放军总医院耳鼻喉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
相关基金：国家自然科学基金面上项目（30572015）、北京市自然科学基金面上项目（7062062）、国家自然科学基金青年科学基金项目（30801285）联合资助

作者：袁永一[1], 黄德亮[1], 韩东一[1], 金政策[2], 戴朴[1]

关键词：耳聋, 线粒体DNA, 实时定量聚合酶链反应, TAQMAN探针, Hearing loss, mtDNA, Real--time PCR, Taqman--based probe

中文摘要：

目的建立以实时荧光定量Taqman探针技术检测线粒体DNA（mitochondial DNA，mtDNA）1494C〉T突变的方法，实现快速、简便、准确筛查这一突变的目标。方法设计针对mtDNA1494C〉T突变的TaqmanMGB探针和引物，从解放军总医院聋病分子诊断中心耳聋DNA库随机选取标本96份，遵循双盲法原则，分别以实时荧光定量TaqmanMGB探针法和序列测定方法检测1494C〉T突变，对检测方法进行可靠性验证。结果实时荧光定量TaqmanMGB探针法检测mtDNA1494C〉T突变，反应时间由原来的12小时缩短到1．5小时，96例耳聋病例中发现mtDNA1494C〉T突变阳性患者11例，阴性85例，与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mtDNA1494C〉T突变的方法简单省时，结果准确直观，特异性强，敏感性高，适用于对母系遗传性聋mtDNA1494C〉T突变的大规模筛查或预防性检查。

英文摘要：

Objective To establish a real--time taqman probe technique to detect the mtDNA 1494C〉T mutation in deaf population. Methods Primers and Taqman MGB probes for mtDNA 1494C〉T mutation were designed and synthesized. Established was the technique system for detecting mtDNA 1494C〉T mutation using realtime taqman probes. 96 patients with severe to profound hearing loss were selected randomly. Based on the double blind principle, mtDNA 1494C〉T mutation was tested by both Taqman MGB probes and sequencing to check the reliability of real-time taqman MGB probes. Results Eleven cases with mtDNA 1494C〉T mutation were found by the technique of real--time taqman probe. These findings coincided with the sequencing results completely. Both the false positive rate and the false negative rate were zero. Conclusion The technique possesses the merits of accuracy, convenience, high sensitivity and specificity, and intuitionistic results. Importantly, the Real--time Taqman probe technique only needs 1.5 hours to detect the 1494C〉T mutation and can save 10.5 hours for one reaction compared with the sequencing method popularly used nowadays. This technique is reliable and suitable for detecting and preventive diagnosis of mtDNA 1494C〉T mutation in a large scale.

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