For instance, molecular methods could detect dead bacteria, or viable but uncultivable bacteria. However, the real-time PCR targeting the atpE gene allows more accurate Mycobacterium spp. quantification, contrary to culture based method which is subjected to many drawbacks such as decontamination artifact (about 2 log10 reduction for M. chelonae), slow mycobacteria growth, clumping of mycobacterial cells, high hydrophobicity of mycobacteria and contamination of culture media by other fast growing environmental microorganisms [44]. Comparison of the method targeting atpE with previously described method targeting 16S rRNA, [17], showed a high correlation. Moreover the
method targeting atpE gene presents two major advantages over the method targeting rrs gene. First, the new method detects all the tested mycobacterial strains, while buy BIX 1294 the method targeting rrs gene
cannot detect isolates of M. Selleckchem AC220 celatum, M. heckeshornense, and M. leprae[17]. Second, the atpE gene is present in a single copy in the Mycobacterium genomes, while the 16S rRNA gene is present either in 1 or 2 copies in the genome [45]. When comparing samples it will be simpler to interpret the data with a stable gene copy number, and probably give a better accuracy of the mycobacterial concentration. One of the limitations of this study is that only 31 mycobacterial species were tested in vitro as positive controls whereas more than 150 mycobacterial species have been described so far [1]. To date, we have confirmed the sensitivity of the atpE real-time PCR method using a large representative collection of mycobacterial species (31 species, e.g. around 20% of described species), including members of MTC (n = 2), M. leprae species (n = 1), slow growing NTM (n = 13), and rapid growing NTM (n = 15). Given the broad diversity of mycobacterial species
we have tested in this study, we expect the method to be applicable to all species within the Mycobacterium genus. In addition, it is the first time that a sensitive and specific molecular target has been identified based on an in silico comparison of 16 mycobacterial (13 species) and 12 non-mycobacterial genomes (4 closely related species). Conclusions In conclusion, Oxaprozin although our strategy did not take into account mTOR inhibitor non-coding regions, such as insertion sequences, repetitive units, non-functional RNA, and structural ribosomal RNAs, the comparison of whole bacterial genomes for design of specific primers is a promising approach not only for mycobacteria but also for other cultured bacterial or archaeal groups for which whole sequenced genomes are accumulating in databases. Metagenomic libraries from environmental samples which are increasingly performed in microbial ecology studies [46] could also provide useful data for the design of specific targets toward uncultured Bacteria and Archaea.