应用重组酶介导的恒温扩增法建立检测非结核分枝杆菌的方法研究

Application of recombination enzyme mediated constant temperature amplification method in detection of non-tuberculous mycobacteria

目的建立一种恒温条件下简便快速的非结核分枝杆菌核酸检测方法,为临床指标提供参考依据。方法设计包括海分枝杆菌、脓肿分枝杆菌和堪萨斯分枝杆菌3种非结核分枝杆菌的特异性引物,采用重组酶介导恒温扩增技术,建立高效快速的非结核分枝杆菌检测方法。结果重组酶介导恒温扩增荧光反应在39℃下进行约20min即可得到检测结果;重组酶介导恒温扩增技术检测3种非结核分枝杆菌的检测下限可达103拷贝/μl,与结核分枝杆菌以及其他临床常见病原菌均无交叉反应,检测特异性为100.0%;对临床相应的非结核分枝杆菌均能很好的检测。结论该方法为非结核分枝杆菌检测提供了一种可在简便环境中特异、灵敏的快速检测手段。

OBJECTIVE To establish a simple and quickly nucleic acid detection method for detecting non-tuberculosis Mycobacterium under the condition of constant temperature,so as to provide reference for clinic.METHODS Specific primers for non-tuberculosis Mycobacterium were designed,including primers for Mycobacterium marinum,Mycobacteriumabscess and Mycobacterium Kansas,and using recombinase-aid amplification（RAA）technology to establish efficient and rapid method for non-tuberculosis Mycobacterium（NTM）detection.RESULTS The whole amplified fluorescence reaction of RAA was completed within 20 min under 39℃.The lowest detection limit of RAA method for detecting three kinds of NTM was 103 copies per microliter,the detection specificity was100%.There were no cross reaction for detecting MTB or other clinical pathogenic bacteria.For clinical corresponding NTM,there were very good positive detection results.CONCLUSIONThe RAA method of NTM detection provides a kind of specific,sensitive and rapid detection in easy environment.