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

摘要

Mycobacteria?are a heterogeneous group of bacteria in terms of their genotypic features and disease association. The accurate and rapid detection of genus?Mycobacterium?andMycobacterium 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?Mycobacteriumand?Mycobacterium tuberculosis?complex (MTC) included the?16SrDNA?and 16S-23S internal transcribed spacer (ITS). Multiplex real time PCR was setup with reference toMycobacterium?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 genusMycobacterium?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 genusMycobacterium?and?Mycobacterium tuberculosis?complex.