【作者】 刘洋； 郭艳玲； 杜博平； 常秀军； 郑晓静； 杜凤娇； 张宗德；

【Author】 Liu Yang,Guo Yanling,Du Boping,et al. Beijing Tuberculosis and Thoracic tumor research institute

【机构】 北京市结核病胸部肿瘤研究所；

【摘要】 目的利用超分支滚环扩增技术对结核分枝杆菌标准株H37Ra进行检测。方法针对结核分枝杆菌复合群特异的插入序列(insert sequence,IS6110)设计锁式探针5’和3’端序列,建立H37Ra的超分支滚环扩增技术检测平台。结果通过优化反应条件,对H37RaIS6110基因的检测灵敏度达到10aM/ml,对H37Ra的菌悬液检测灵敏度为10cfu/ml。而对胞内分枝杆菌和肺炎链球菌的检测结果为阴性。结论试验证明利用超分支滚环扩增技术可以灵敏、特异的检测H37Ra,超分支滚环扩增技术检测结核分枝杆菌基因体系的建立,有助于建立一种简单实用,结果可靠的结核菌新技术检测平台。更多还原

【Abstract】 Objective A highly sensitive,specific method,Hyperbranched Rolling circle amplification(HRCA),was optimized and used to detect the standard strain H37Ra of M. tuberculosis.Methods The sequences of the 5’ terminal and 3’ terminal of the padlock probe was designed according to the sequences of Insert Sequence 6110(IS6110),which was specific for the Mycobacterium tuberculosis complex.The padlock probe was used in HRCA to detect the sequence of IS6110 in H37Ra.Results Through the optimization of HRCA,the sensitivity of HRCA in detection of purified DNA from H37Ra was 10 aM/ml,and the sensitivity in detection of H37Ra culture suspension was 10 cfu/ml.For testing the specificity of HRCA,the padlock probe was used to detect Streptococcus pneumoniae and Mycobacterium intercellulare,the result was negative.Conclusions The HRCA technique can be considered as a rapid and accurate method to detect H37Ra.The establishment of HRCA in detection of specific gene of H37Ra is useful for development of a cost-effective and reliable method in tuberculosis diagnosis.更多还原