title: 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.
authors.affiliation: Mie Prefecture Health and Environment Research Institute, 3684-11 Sakura, Yokkaichi, Mie, 512-1211, Japan, nagaiy02@pref.mie.jp.
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Nagai
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Iwade
authors.forename: Eri
authors.initials: E
authors.lastname: Hayakawa
authors.forename: Manabu
authors.initials: M
authors.lastname: Nakano
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mitarai
authors.forename: Masahiko
authors.initials: M
authors.lastname: Katayama
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nosaka
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamaguchi
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial