Key Laboratory of Medical Molecular Virology, Institutes of Biomedical Sciences and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China.
J Clin Microbiol. 2011 Sep;49(9):3132-8. doi: 10.1128/JCM.02046-10. Epub 2011 Jul 13.
Early diagnosis of drug-resistant Mycobacterium tuberculosis is urgently needed to optimize treatment regimens and to prevent the transmission of resistant strains. Real-time PCR assays have been developed to detect drug resistance rapidly, but none of them have been widely applied due to their complexity, high cost, or requirement for advanced instruments. In this study, we developed a real-time PCR method based on melting curve analysis of dually labeled probes. Six probes targeting the rpoB 81-bp core region, katG315, the inhA promoter, the ahpC promoter, and embB306 were designed and validated with clinical isolates. First, 10 multidrug-resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (T(m)) deviations of different mutations by single real-time PCR. All mutations can be detected by significant T(m) reductions compared to the wild type. Then, three duplex real-time PCRs, with two probes in each, were developed to detect mutations in 158 MDR isolates. Comparison of the results with the sequencing data showed that all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug-resistant mutations in M. tuberculosis. Compared to other real-time PCR methods, we use fewer probes, which are labeled with the same fluorophore, guaranteeing that this assay can be used for detection in a single fluorescent channel or can be run on single-channel instruments. In conclusion, we have developed a widely applicable real-time PCR assay to detect drug-resistant mutations in M. tuberculosis.
早期诊断耐药结核分枝杆菌对于优化治疗方案和防止耐药菌株传播至关重要。已经开发了实时 PCR 检测方法来快速检测耐药性，但由于其复杂性、高成本或对先进仪器的要求，这些方法均未得到广泛应用。在这项研究中，我们开发了一种基于双重标记探针熔解曲线分析的实时 PCR 方法。设计并验证了针对 rpoB 81-bp 核心区域、katG315、inhA 启动子、ahpC 启动子和 embB306 的 6 个探针，并用临床分离株进行了分析。首先，用 10 株具有广泛突变谱的多药耐药（MDR）菌株通过单重实时 PCR 分析不同突变的熔解温度（T(m))偏差。与野生型相比，所有突变均导致 T(m)显著降低，从而可以检测到所有突变。然后，开发了三种双链实时 PCR，每个反应包含两个探针，用于检测 158 株 MDR 分离株中的突变。与测序数据的比较表明，用六个探针检测到的所有突变均具有 100%的敏感性和 100%的特异性。我们的方法为快速检测结核分枝杆菌中的耐药突变提供了一种新方法。与其他实时 PCR 方法相比，我们使用了更少的探针，这些探针用相同的荧光基团标记，保证了该检测可以在单个荧光通道中进行，或者可以在单通道仪器上运行。总之，我们开发了一种广泛适用的实时 PCR 检测方法，用于检测结核分枝杆菌中的耐药突变。
