Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis by High-Resolution Melting Analysis

摘要

We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position ?15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.