FISHing Mycobacterium tuberculosis Complex by Use of a rpoB DNA Probe Bait

Routine staining of sputum specimens does not identify acid-fast bacilli as Mycobacterium tuberculosis with utmost precision, limiting its usability as a confirmatory test for pulmonary tuberculosis. We have combined Ziehl-Neelsen staining and fluorescence in situ hybridization (FISH) to detect M. tuberculosis in sputum specimens. We have developed a new fluorescent oligonucleotide rpoBMTC probe (5'-Alexa-555-AGCGGGGTGATGTCAACCCAG-3') targeting the M. tuberculosis complex rpoB gene. In silico alignment yielded 100% match for M. tuberculosis complex mycobacteria, 66.6% to 47.6% for other bacteria, and no significant hits for viruses and eukaryotes. Negative binding of rpoBMTC probe to the top six respiratory tract bacterial pathogens and to Mycobacterium abscessus and Mycobacterium avium experimentally confirmed its specificity. As for sensitivity, rpoBMTC-FISH detected 10³ CFU/ml M. tuberculosis as confirmed by successful detection of M. tuberculosis in artificially seeded sputum samples. The application of rpoBMTC-FISH to 116 routine sputum specimens yielded a detection of M. tuberculosis in all of the 31 Ziehl-Neelsen-positive and culture-positive specimens, and no detection of M. tuberculosis in the 85 M. tuberculosis-negative specimens. These data established the proof of concept that rpoBMTC-FISH alone or combined with Ziehl-Neelsen staining can specifically "FISH out" M. tuberculosis complex mycobacteria in sputum samples collected from patients suspected of pulmonary mycobacteriosis. We are implementing this probe for the routine and specific detection of M. tuberculosis complex bacteria in sputum exhibiting acid-fast mycobacteria.