Rapid-Cycle PCR and Fluorimetry for Detection of Mycobacteria

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

LightCycler Technology in Molecular Diagnostics

“…For example, dual-hybridization probes have been used to detect 33 species of mycobacteria using the 16S rRNA gene. 75 LightCycler technology has been applied to many bacteria, fungi, and parasites. Targeting a single pathogen of clinical importance can immediately guide therapy.…”

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

“…Many regions of the mycobacterial genome have been used in molecular diagnosis of TB, such as IS6110, 16S rRNA, hsp65, rpoB, sdaA, devR and mpt64 (Manjunath et al, 1991;Lachnik et al, 2002;Kim et al, 2004;Chakravorty et al, 2005;Negi et al, 2007;Hwang et al, 2013;Nimesh et al, 2013). mpt64 is found as a single copy in the genome of MTBC species (Lee et al, 1994), and has been used for both pulmonary and extrapulmonary TB diagnosis (Tan et al, 1995;Dar et al, 1998;Martins et al, 2000;Bhanu et al, 2005;Therese et al, 2005;Takahashi & Nakayama, 2006;Dil-Afroze et al, 2008;Kusum et al, 2012;Sethi et al, 2012).…”

Detection of Mycobacterium tuberculosis by Real-Time PCR Using Pan-Mycobacterial Primers and a Pair of Fluorescence Resonance Energy Transfer Probes Specific for the M. tuberculosis Complex

“…Building on this work, many groups have shown that drug response is also influenced by genomic variation (6 ). Multiple SNPs have been identified that have a major impact on response to chemotherapy (7)(8)(9)(10)(11); it is therefore necessary to have rapid and efficient SNP evaluation techniques to analyze genes that influence chemotherapy response.…”