荧光置换探针多重实时PCR检测结核杆菌3种耐药突变

目的]建立单管检测结核分枝杆菌3种耐药突变的多重实时PCR检测方法,考察其敏感性并应用于痰标本的检测。方法]将荧光双链置换探针、实时PCR和多重PCR相结合,针对3种一线药物链霉素、异烟肼和乙胺丁醇中最常见的且都是由于点突变而导致的耐药突变位点进行检测。结果]该检测体系可以检测到5个菌/test。用该体系检测9份已知基因型的结核分枝杆菌培养标本和118份痰标本,所有检测结果均通过ARMS体系验证。结论]该检测体系具有很高的敏感性和特异性,检测快速,测定突变位点范围较其它技术有较大提高,且操作简便,有利于结核病菌的早期耐药检测和指导医生用药。

Multiple Real-time PCR Detection of 3 Drug Resistance Mutations in Mycobacterium Tuberculosis with Displacing Probes

Objective] To establish PCR assay and evaluate the feasibility of displacing probes and detect related resistance mutations in DNA extracts from sputum samples.Method] Displacing probes,real-time PCR and multiplex PCR technology were used and 3 genes(rpsL,katG and embB) associated with streptomycin,isoniazid and ethambutol resistance were detected respectively.Results] Each test of 5 copies mycobacterium tuberculosis can be detected and 9 mycobacterium tuberculosis isolates and 118 sputum samples were evaluated with this assay.All results of real-time PCR assay were consistent with the ARMS assay.Conclusions] Comparing with classical methods,this assay is of rapid,high sensitivity and specificity.It can be used for high-throughout screening for the mutations of drug resistance in mycobacterium tuberculosis.