Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply “M. bovis”]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide −215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG

Rapid-Cycle PCR and Fluorimetry for Detection of Mycobacteria

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 culture-positive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory