Fast identification of Mycobacteria from positive Mb/Bact bottles using a multiplex PCR

Background: The rapid identification of mycobacteria from culture-positive Mb/Bact bottles is an important clinical issue. Furthermore, the availability of a non-expensive, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In this study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from positive Mb/Bact (Organon-Teknika) bottles. A multiplex PCR focusing the hsp65 gene, and the IS6110 was done and evaluated for its efficiency compared to PRA identfication, and PNB inhibition. Methods: Mycobacterial strains were grown from clinical samples and preserved in solid media (LJ). A panel of 91 M. tuberculosis strains, and 26 Non-TuberculousMycobacteria were submitted to the following identification techniques: a) hsp65 gene amplification followed by restriction (PRA); b) p-Nitrobenzoic acid (PNB) growth inhibition in liquid media (Mb/Bact); 3) Multiplex-PCR, using two sets of primers: TB284/TB850 (IS6110 MTC spcific primers), and TB11/TB12 (65 kD antigen, Mycobacterium genus specific primers). The mycobacteria was inactivated being heated at 80oC for 20min before DNA extraction. DNA was extracted by the freezing and thawing method. 50 l of the the supernatant was used for amplification. Identification using hsp65 restriction was done according to international protocols (Telenti et al, 1993), and considered the gold-standard. PNB was added to liquid Mb/Bact bottles to a final concentration of 500 g/ml. p-aminobenzoic acid, and the Mycobacterial concentration was adjusted to the MacFarland 1 standard. Results: The hsp65 restriction analysis identified all Mycobacteria tested as: M.tuberculosis (61 strains), M.avium, M.intracellulare, M.fortuitum, M.abscessus, M.gordonae, M.szulgai. There was 100% agreement between the tests performed Conclusion: A simple and inexpensive Multiplex-PCR as described here, can readly distinguish among M. tuberculosis Complex strains, and Non-Tuberculous Mycobacteria.