High resolution melting curve assay for rapid detection of drug-resistant Mycobacterium tuberculosis

Abstract We developed and evaluated a high resolution melting (HRM) curve assay by using real-time PCR for the detection of the most frequent mutations of Mycobacterium tuberculosis , which are responsible for the resistance of four anti-TB drugs: rifampicin, isoniazid, ethambutol, and streptomycin. The HRM assay was successfully used for the detection of dominant mutations: A516V, H526A, H526T, S531L, L533P, and A516G/S531L in rpoB ; S315T, and S315A in katG ; −15C/T, and −8T/C in mab - inhA ; M306I in embB ; K88Q and K43R in rpsL ; and 513A/C in rrs . We were able to discriminate the mutant from the wild type by analyzing the melting-curve shape in 40 clinical M. tuberculosis isolates, and the results of the HRM assay were completely consistent with those of DNA sequencing. This HRM assay is a simple, rapid, and cost-effective method that can be performed in a closed tube. Therefore, our assay is a potentially useful tool for the rapid detection of drug-resistant M. tuberculosis.