结核分枝杆菌实时荧光核酸恒温扩增检测方法的建立及应用

Establishment and application of simultaneous amplification and testing assay for detection of Mycobacterium tuberculosis

目的建立结核分枝杆菌实时荧光核酸恒温扩增检测方法(SAT-TB),并探讨其在结核病诊断中的应用。方法设计特异性引物和探针,构建SAT检测体系,并优化。通过不同浓度H37Rv标准菌株检测评估其灵敏性。结果结核分枝杆菌SAT检测灵敏度为10CFU/ml;非结核分枝杆菌及常规细菌未检出TB RNA。肺结核患者痰液标本中结核分枝杆菌SAT检出率为54%,显著高于痰培养法的40%(P<0.05)及痰涂片法的34%(P<0.01)。结论 SAT方法应用于结核分枝杆菌检测,快速方便、灵敏度高、特异性强,可以应用于临床结核病诊断。

Objective To establish and apply the simultaneous amplification and testing（SAT） for detecting and diagnosing Mycobacterium tuberculosis.Methods The specific primers and probes were designed to establish and optimize the SAT system.The sensitivity was analyzed by detecting different concentrations of H37Rv with SAT.Results The sensitivity of SAT assay for detecting H37Rv was 10 CFU/ml.TB-RNA was not detected in the other non-tuberculous mycobacteria and common bacteria.The positive rate of SAT assay was 54% in clinical samples from suspected tuberculosis cases,which was much higher than 40% by sputum culture（P0.05） and 34% by sputum smear（P0.01）.Conclusion SAT assay for detecting mycobacteria can be used for the diagnosis of tuberculosis with the advantages of being sensitive,specific,fast and simple.