聚合酶螺旋反应双指示法检测结核分枝杆菌的研究  

Mycobacterium tuberculosis detection with polymerase spiral reaction and dual indicators

作　　者：施琦 刘威 张乔 贺晓明 慈颖[1] 邹大阳 张永江[1] 王静[1] SHI Qi;LIU Wei;ZHANG Qiao;HE Xiao-ming;CI Ying;ZOU Da-yang;ZHANG Yong-jiang;WANG Jing(Institute of Health Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;不详)

机构地区：[1]中国检验检疫科学研究院卫检所,北京100176 [2]中国人民解放军疾病预防控制中心

出　　处：《中国国境卫生检疫杂志》2021年第2期77-80,共4页Chinese Journal of Frontier Health and Quarantine

基　　金：国家重点研发计划项目(2016YFF0203203)。

摘　　要：目的建立聚合酶螺旋反应双指示法检测结核分枝杆菌,满足现场监测和快速检测的需求。方法基于结核分枝杆菌聚合酶螺旋反应体系,利用Bst DNA聚合酶链置换活性,在同一反应体系中加入核酸荧光染料EveGreen和pH显色剂两种指示物,用实时荧光法和可视显色法同时判断反应结果,用于痰样本中结核分枝杆菌的检测。结果建立的双指示法对结核分枝杆菌核酸最低检测浓度均为10 pg/μl,可在65 min内得到结果,敏感性和特异性良好,与金黄色葡萄球菌、福氏志贺菌、大肠杆菌、铜绿假单胞菌、肺炎克雷伯菌均无交叉反应。结论建立的聚合酶螺旋反应双指示检测法为结核分枝杆菌的快速检测提供了新方法。Objective To establish a method based on polymerase spiral reaction and dual indicators for the detection of Mycobacterium tuberculosis that meets the requirements of on-site monitoring and rapid detection.Methods The detection was based on the polymerase spiral reaction system of M.tuberculosis and the chain displacement activity of Bst DNA polymerase.The nucleic acid fluorescent dye EveGreen and pH indicators were added to the re-action system.The reaction was monitored by using real-time fluorescence and visual inspection for the detection of M.tuberculosis in sputum samples.Results With the established method,the minimum detection limit of M.tuberculosis nucleic acid was 10 pg/μl,and the detection time was <65 min.The sensitivity and specificity were satisfactory,and there were no cross reactions with Staphylococcus aureus,Shigella flexneri,Escherichia coli,Pseudomonas aeruginosa,and Klebsiella pneumoniae.Conclusion The method based on polymerase spiral reaction and dual indicators provided a new means for the rapid detection of M.tuberculosis.

关 键 词：聚合酶螺旋反应 双指示剂 结核分枝杆菌 

分 类 号：R183.3[医药卫生—流行病学]