Multiplex PCR Assay for Identification of Mycobacterial Species Isolated from Liquid Cultures

We developed a 2-step multiplex polymerase chain reaction (PCR) assay to rapidly identify the most common mycobacterial species isolated from positive liquid cultures as follows. In the first step, a multiplex PCR-A assay was used to differentiate Mycobacterium tuberculosis complex from nontuberculous myco-bacterial (NTM) species. In the second step, 2 parallel multiplex PCR-B/C assays were used to identify M. avium, M. intracellulare, M. kansasii, M. fortuitum, M. abscessus/chelonae, M. gordonae, and M. terrae. The multiplex PCR assays were tested on a total of 147 liquid cultures, including 15 reference strains and 4 mixed cultures. The results were compared with those obtained by PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and species-specific PCR. For 129 of 143 cultures yielding a single isolate, the results of the multiplex PCR matched those of species-specific PCR and PRA-hsp65 except for 8 M. intracellulare isolates and 6 other NTM species not represented by the multiplex PCR in this study. Moreover, the multiplex PCR detected the coexistence of 2 mycobacterial species in 4 mixed cultures, whereas PRA-hsp65 misidentified the mixed cultures as a single species. The data demonstrated that multi-plex PCR assays may be easy and useful for the rapid identification of most common mycobacterial spe-cies in liquid cultures, particularly in mixed cultures.