Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7

Abstract

Identification ofMycobacterium tuberculosisandM. bovisis necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection ofM. tuberculosisandM. bovis.As a BLAST and BLASTP search we selected regulatory genewhiB7that encodes multi-drug resistance in this bacterium. Thirty clinical isolates ofM. tuberculosiswere sequenced and all the mutations in genewhiB7were detected. The best set of several pairs of primers was selected and used in comparison byrpoBgene for differentiation ofM. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenicMycobacteriumisolates and 30 clinical isolates ofM. tuberculosis.It was proved that only clinical isolates ofM. tuberculosisandM. bovishave positive bands of 667 bpwhiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products ofwhiB7gene were observed for ten positive sputum samples (preliminarily approved to be positive forM. tuberculosisby commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoBgene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification ofM. tuberculosisandM. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.