In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium, and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria, M. tuberculosis complex, and M. avium were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the M. tuberculosis complex-specific probe and the M. avium-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified M. tuberculosis complex and M. avium without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culturepositive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.
Mycobacterium tuberculosis, the major pathogen of human tuberculosis, predominantly affects the respiratory tract, whereas M. bovis, the major pathogen of tuberculosis in cattle, is typically found in extrapulmonary tuberculosis (5, 32). Attenuated M. bovis BCG, the only currently available vaccine against tuberculosis, has been administered to more than 3 billion people worldwide (17). It may be isolated from immunocompromised individuals, who might develop disseminated disease with the vaccine strain after vaccination with M. bovis BCG. M. africanum and M. microti are rarely encountered members of the M. tuberculosis complex (MTBC) (11,18,22). M. africanum causes human tuberculosis in certain regions of tropical Africa. M. microti causes naturally acquired generalized tuberculosis in voles and produces local lesions in guinea pigs, rabbits, and calves and has been described very occasionally as a cause of infection in human immunodeficiency virus-positive patients.Nitrate reductase activity is a widely used phenotypic trait to differentiate between M. tuberculosis, which rapidly accumulates nitrite from nitrate, and other members of the MTBC (18). In other pathogenic and environmental bacteria, at least three different nitrate reductases have been found, one of which is the respiratory nitrate reductase encoded by narGHJI (19). The membrane-bound complex consists of NarG, -H, and -I, with NarG being the catalytic subunit, whereas NarJ is required for the assembly of the enzyme. Expression of narGHJI is typically induced under anaerobic conditions (20). Previous studies on mycobacterial nitrate reduction were limited to its role in classification and identification of the genus Mycobacterium (2, 6, 28, 29). However, recent reports revived interest in a possible role for enzymes involved in nitrate metabolism during infection with M. tuberculosis or M. bovis BCG (4, 12, 30). We provided evidence that M. bovis BCG weakly accumulates nitrite from nitrate under strictly anaerobic conditions, that this activity is mediated by narGHJI, and that a mutant of M. bovis BCG with a partial deletion of the narG gene was attenuated in mice, linking anaerobic nitrate reduction to mycobacterial pathogenesis (7,31).In the present study, we generated targeted deletion of narG to compare the role of narGHJI in nitrite accumulation by M. tuberculosis, M. bovis (non-BCG [referred to here as simply "M. bovis"]), and M. bovis BCG. Analysis of the promoter region of narGHJI from either species revealed a single nucleotide polymorphism that separated M. tuberculosis from the bovine mycobacteria. We tested a variety of different strains within the MTBC by using LightCycler technology and showed that this single nucleotide polymorphism was specific for M. tuberculosis, thus allowing rapid identification of M. tuberculosis. For further differentiation within the MTBC based on LightCycler technology, we adapted analysis of the oxyR polymorphism, which is specific for the bovine mycobacteria, and the region of differences 1 (RD1) polymorphi...
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