[Detection and identification of mycobacteria by PCR-RFLP method]

The frequency of isolation of Mycobacterium tuberculosis has reported to be decreased, however, Mycobacterium species including MOTT (Mycobacteria other than M. tuberculosis complex) still remain to be the important bacteria causing opportunistic infection. Two clinical procedures have long been adopted to date for the detection of acid fast bacteria belonging to the family Mycobacteriaceae. One is the direct microscopic examination of the specimen stained by Ziehl-Neelsen method, which is rapid, but is relatively insensitive and less specific. And another is a time- consuming culturing technique on slants of Ogawa's media, which takes 4 to 8 weeks of incubation. Recently, various methods to detect mycobacterial DNA have been reported. However, they require a complicated and tedious processes. We represent here a simple method for the detection and identification of Mycobacteria (M. tuberculosis, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium chelonae) using PCR-RFLP method for the 65kDa antigen with high sensitivity and specificity. This includes the amplification of Mycobacterial gene encoding a part of the 65kDa antigen. Subsequently, we sequenced the region amplified by PCR from the seven standard bacterial strains. As the results, we found that HaeIII restriction enzyme is suitable for the prompt and easy discrimination among the seven strains examined. These findings led us to conclude that this method is time-saving and possibly applicable for the rapid detection of Mycobacterial species including MOTT from clinical specimens in routine clinical laboratories.