Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB)

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which co mprises the Rif(r) region, was used for the differential identification of 49 mycobacteria, The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al,, J, Clin, Microbiol. 37:1714-1720, 1999), Digestion with four restriction en zymes (Haem, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not onl y the reference strains but also the clinical isolates of mycobacteria to b e distinguished. Both rapidly and slowly growing mycobacteria were distinct ly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria, Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, a nd other closely related species, were distinguished by simultaneous digest ion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of cli nical isolates could be identified, It was also possible to detect and iden tify M, tuberculosis directly from sputa and bronchoalveolar lavage specime ns. These results suggest that PRA of rpoB DNA is a simple and feasible met hod not only for the differentiation of culture isolates but also for the r apid detection and identification of pathogenic mycobacteria in primary cli nical specimens.