JLS (NP), Mycobacterium sp. KMS (NP), Mycobacterium sp. MCS (NP), M. ulcerans (P), M. vanbaalenii (NP), [24–26]. Moreover, three whole genomes of other NTM species were sequenced and are currently assembled (M. intracellulare, M. kansasii, M. parascrofulaceum). This increasing number of completely sequenced mycobacterial genomes led to the development of the MycoHit software, which permits gene- and protein-level comparisons across mycobacteria species, . This software was originally developed to detect horizontal gene transfers and mutations among whole mycobacterial genomes . However, MycoHit selleck products should also be useful for developing new primers
and probes for mycobacteria detection and quantification in environmental and clinical samples. In this paper, we used this tool for screening sensitive and specific targets of Mycobacterium spp.. We compared in silico proteins of whole mycobacterial genomes with those of non-mycobacterial genomes using the MycoHit software, in order to find conserved sequences among mycobacteria that will not be shared with non-mycobacterial species. Based on the screening results a primer pair and a probe targeting the atpE gene were designed and tested by real-time PCR. This novel target proved to be totally specific and sensitive. It also offers the advantage of targeting a gene present as a single copy in the
genome. Thus this new real-time PCR method appears promising for water quality survey, and should be useful for studying the ecology of mycobacteria in aquatic, terrestrial Microtubule Associated inhibitor and urban environments. Results Specificity of genes commonly used for mycobacterial detection/identification Excluding rrs gene and ITS (non-functional RNA
elements and structural ribosomal RNAs), and according to our strategy of genome comparison (Figure 1) most of the genes commonly used for mycobacterial species identification (gyrA, gyrB, hsp65, recA, rpoB, sodA, groEL1, groEL2) code for proteins which present similar see more conformations in non-mycobacterial studied genomes (Additional file 1). Indeed, protein similarity levels of these genes, in comparison with M. Geneticin manufacturer tuberculosis H37Rv genome, were higher than 80% for the other 15 mycobacterial genomes studied (96 ± 2% for gyrA, 94 ± 5% for gyrB, 79 ± 5% for groEL1, 93 ± 4% for groEL2 which is an alternative gene name for hsp65, 99 ± 1% for recA, 96 ± 2% for rpoB, 81 ± 33% for sodA), and also for the 12 non-mycobacterial genomes studied (86 ± 5% for gyrA, 85 ± 5% for gyrB, 89 ± 3% for groEL1, 96 ± 2% for groEL2, 94 ± 3% for recA, 88 ± 4% for rpoB, 69 ± 22% for sodA). Figure 1 Strategy used to identify sensitive and specific targets in Mycobacterium spp. whole genomes based on MycoHit software. DNA sequences of targeted mycobacterial genomes include M. tuberculosis H37Ra (CP000611.1), M. tuberculosis CDC 1551 (AE000516.2), M. tuberculosis KZN 1435 (CP001658.1), M. bovis AF2122/97 (BX248333.1), M. ulcerans Agy99 (CP000325.1), M. marinum M (CP000854.1), M. avium 104 (CP000479.