FISHing Mycobacterium tuberculosis Complex by Use of a <i>rpoB</i> DNA Probe Bait

Abstract

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