BACKGROUND: Recently the clinical significance of several mycobacterial species has been increased and there is a growing need to identify mycobacteria to the species level. We evaluated multiplex polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assay for identification of mycobacterial isolates. METHODS: Reference strains of 6 species of mycobacteria and 88 clinical isolates were lysed by boiling method. The lysates were used for multiplex PCR reactions incorporating three pairs of PCR primers, which were expected to amplify fragments from the 65-kDa gene common to all mycobacteria, genes of M. tuberculosis complex and M. avium, respectively. The resultant amplicons were digested with the restric-tion enzymes PspEI and HaeIII. Multiplex PCR products and digested products were visualized by electrophoresis on agarose gels. RESULTS: Six reference strains yielded compatible results. Eighty-eight clinical isolates were identified as M. tuberculosis complex (81 strains), M. avium (2 strains), M. intracellulare (2 strains), M. fortuitum biovariant peregrinum (2 strains), and M. gordonae III (1 strain). CONCLUSION: Multiplex PCR-RFLP assay appears to be a reliable method for rapid identification of mycobacteria to species level.
Electrophoresis ; Gels ; Gordonia Bacterium ; Multiplex Polymerase Chain Reaction ; Mycobacterium* ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sepharose ; Tuberculosis