title: Rapid detection of pyrazinamide resistant Mycobacterium tuberculosis by high resolution melting curve analysis.
abstract: OBJECTIVE
  To develop a high-resolution melting curve analysis (HRM) assay for detection of PZA resistance.
  
  
  METHODS
  Thirty samples of PZA-susceptible M. tuberculosis and eight isolates of PZA-resistant M. tuberculosis were included in the experiment. Five sets of primers were designed to cover the pncA gene and its upstream nucleotides. The pncA gene fragments were amplified by the PCR method. Determination of pncA mutation in the sample by comparing their melting behavior of the PCR products with the M. tuberculosis wild type by using Gene scanning software of the LightCycler 480 instrument.
  
  
  RESULTS
  Mutations were clearly detected in all PZA resistant samples by the HRM, whereas all PZA susceptible samples showed no mutation in the pncA gene. Results were concordant with the drug susceptibility testing by using BACTEC MGIT 960 PZA kit and mutation detection by the DNA sequencing method.
  
  
  CONCLUSIONS
  This HRM method offers a rapid and reliable screen for PZA resistant M. tuberculosis.
authors.affiliation: Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Worawan
authors.initials: W
authors.lastname: Watcharasamphankul
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
authors.forename: Suporn
authors.initials: S
authors.lastname: Foongladda
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011718:Pyrazinamide / Q000494:pharmacology*; D044366:Transition Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000188:drug therapy*
chemical_list: D000995:Antitubercular Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D011718:Pyrazinamide