atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples
- Equal contributors
1 Laboratoire Eau Environnement Systèmes Urbains (Leesu) UMR MA 102-AgroParisTech, Université Paris-Est, 6-8 avenue Blaise Pascal Cité, Descartes, FR 77455, Champs sur Marne, France
2 Research Institute of the McGill University Health Centre, 1650 Cedar Avenue, Montreal H3G 1A4, QC, Canada
3 Département Infection et Epidémiologie, Infections Bactériennes Invasives, Institut Pasteur, 28 Rue du Dr. Roux, Paris, F 75015, France
4 Laboratoire associé du Centre national de référence des mycobactéries et de la résistance aux antituberculeux, AP-HP, Groupe Hospitalier Saint Louis-Lariboisière, Université Paris Diderot EA3964, FR 75475, Paris, France
5 Eau de Paris, Direction Recherche et Développement Qualité de l'Eau (DRDQE), 33 avenue Jean Jaurès, FR 94200, Ivry-sur-Seine, France
BMC Microbiology 2013, 13:277 doi:10.1186/1471-2180-13-277Published: 3 December 2013
The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets.
Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.
The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.