A novel multiplex real time PCR for rapid and accurate identification of the genus Mycobacterium and Mycobacterium tuberculosis complex species

Mycobacteria are a heterogeneous group of bacteria in terms of their genotypic features and disease association. The accurate and rapid detection of genus Mycobacterium and Mycobacterium tuberculosis complex (MTC) isolates from primary culture is important due to timely and appropriate antibiotic therapy. In this study, we developed a novel multiplex real time PCR for rapid identification of Mycobacteria from other genus of bacteria and Mycobacterium tuberculosis complex from non-tuberculosis Mycobacteria (NTMs). Genetic targets for primer designing and identification of genus Mycobacterium and Mycobacterium tuberculosis complex (MTC) included the 16SrDNA and 16S-23S internal transcribed spacer (ITS). Multiplex real time PCR was setup with reference to Mycobacterium strains. Results of real time PCR were analyzed with melting curves and determined melting temperature of genus Mycobacterium and Mycobacterium tuberculosis complex. For the assessment of the sensitivity and specificity of technique, fifty three clinical isolates of Mycobacteria were collected from different Tuberculousis Centers in Iran. Multiplex real time PCR results were compared by PCR-based sequencing method with amplification and sequencing of 16S-23S ribosomal RNA internal transcribed spacer (ITS) for identification of Mycobacterium species. Of 53 isolates, 52 (98%) isolates identified as genus Mycobacterium and 34 isolates as MTC (65%). Sensitivity and specificity of multiplex real time PCR were 96%, 100% for genus Mycobacterium and 100%, 100% for Mycobacterium tuberculosis complex (MTC), respectively. The multiplex real time PCR with melting curve analysis in the present study is a reliable and suitable method for rapid and accurate identification of genus Mycobacterium and Mycobacterium tuberculosis complex.