Rapid Detection of Pyrazinamide Resistant Mycobacterium tuberculosis by High Resolution Melting Curve Analysis
Worawan Watcharasamphankul MSc*, Eric R Houpt MD**, Suporn Foongladda DVM, PhD*
Affiliation : * Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand ** Department of Internal Medicine, Faculty of Medicine, University of Virginia, USA
Objective : To develop a high-resolution melting curve analysis (HRM) assay for detection of PZA resistance.
Material and Method: 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.
Conclusion : This HRM method offers a rapid and reliable screen for PZA resistant M. tuberculosis.
Keywords : Mycobacterium, Pyrazinamide, Drug resistant, pncA gene, High resolution melting curve analysis
