Nevertheless, PE and PPE family proteins, and proteins coded by esx gene clusters are very small and polymorphous among genomes of the 11 NTM species compared (Table 1). Mycobacterial cell wall is also important in pathology, and could procure interesting PCR targets. For instance, several studies emphasized that cyclopropanation of the mycolic acids is common among pathogenic mycobacteria but rare
among saprophytic species . Although having sufficient length, proteins CMAS coded by the cmaA1 gene and lipoprotein coded by lppM gene in M. tuberculosis H37Rv, were also polymorphous among genomes of the 11 NTM species compared PF-02341066 manufacturer (Table 1) and thus could not be used to design a primer pair and a probe (Additional file 2). Nevertheless, polymorphism of mycobacterial mycolic acids is useful for mycobacteria identification [40, 41]. The atpE gene which codes ATP synthase subunit C in M. tuberculosis H37Rv genome (locus Rv1305) is exclusively conserved in the genomes of the 17 mycobacterial species studied (Additional file 2), and its length and relative conservation among mycobacteria make it an adequate molecular target in order to detect Mycobacterium genus. It is remarkable to see that the protein coded by atpE gene was also click here the target of the new antimycobacterial compound recently
described: diarylquinoline R207910 . This compound shows a specific bactericidal effect on mycobacteria and none in other genera . In addition, our in Selleck ZD1839 vitro results demonstrated the specificity of the atpE gene (locus Rv1305), which codes for the ATP synthase protein subunit C. These results also showed that our strategy of target design based on MycoHit software (Figure 1) gave very useful results for designing highly specific primers and might be applied to other microorganism clusters. In vitro validation of the real-time PCR targeting the atpE gene showed a very high specificity and sensitivity, as well as reproducible
quantification of different mycobacteria species. The new real-time method was tested on a realistic number of mycobacterial species including several slow and rapid growing NTM, although not all the described mycobacterial species were tested. In addition, application of this real-time PCR method to environmental samples showed that Mycobacterium was detected in tap water samples. The discrepancy between the cultural and molecular techniques was previously described for other pathogens, and the lower level of prevalence obtained by the PCR methods was probably due to our concentration and extraction procedures. These protocol steps must be improved to detect low level of NTM even if the used spin column seemed more appropriate for DNA extraction from environmental samples compared to classical phenol-chloroform extraction. Moreover, culture method did not detect higher level of mycobacterial cells compared to the molecular one.