Rapid-Cycle PCR and Fluorimetry for Detection of Mycobacteria

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Abstract
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.