Detection of rpoB, inhA and katG Genes Mutations in Clinical Isolates of Mycobacterium tuberculosis by Real-Time PCR Based on Taqman and HRM Assays

The rapid identification of patients carrying resistant Mycobacterium tuberculosis (M.TB) isolates is important for effective tuberculosis therapy. Unfortunately, during the recent years considerable numbers of isolates showed resistant to Rifampin (RIF) and Isoniazid (INH). The aim of this study was to rapidly identify resistant MTB isolates using molecular method. For this reason, the comparison between real-time PCR based on Taqman and HRM AssayS in detection of rpoB, inhA and katG genes mutation in clinical isolates were performed and analyzed. The study carried out on Mycobacteriology Research Center (MRC) from 2012-2013. Classical susceptibility testing i.e., proportional method against INH and RIF was performed on eighty three M.TB isolates. Thereafter, multiplex and real-time PCR were performed on extracted DNA sample. The real-time PCR was based on Taqman and HRM assays. Mutation in genes rpoB, inhA and katG were detected. In overall, based on proportional and multiplex PCR method, 47 and 35 isolates were resistant to RIF and INH, respectively. Thirty of strains were resistant to both RIF and INH. The agreement of real-time PCR using Taqman was 88% for resistant and 84% for susceptible isolates, whereas the agreement of HRM was 96% and 30%, respectively. The sensitivity and specificity of Taqman in comparison to multiplex were 84% and 88%, respectively. In addition, the sensitivity and specificity of HRM were 30% and 96%, respectively. Results documented that real-time PCR based on Taqman assay is more sensitive than HRM assay. Additionally, real-time PCR based on Taqman assay is a rapid, accurate and cost effective method in detection of Mycobacterium tuberculosis resistance.