title: High-resolution melting curve analysis for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates.
abstract: We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position -15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.
authors.affiliation: Department of Laboratory Medicine, School of Medicine, Pusan National University, Busan, South Korea.
authors.forename: Go Eun
authors.initials: GE
authors.lastname: Choi
authors.forename: Sun Min
authors.initials: SM
authors.lastname: Lee
authors.forename: Jongyoun
authors.initials: J
authors.lastname: Yi
authors.forename: Sang Hyun
authors.initials: SH
authors.lastname: Hwang
authors.forename: Hyung Hoi
authors.initials: HH
authors.lastname: Kim
authors.forename: Eun Yup
authors.initials: EY
authors.lastname: Lee
authors.forename: Eun Hae
authors.initials: EH
authors.lastname: Cho
authors.forename: Jee Hee
authors.initials: JH
authors.lastname: Kim
authors.forename: Hwa-Jung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Chulhun L
authors.initials: CL
authors.lastname: Chang
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D007538:Isoniazid; D012293:Rifampin