Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Dec 3:13:277.
doi: 10.1186/1471-2180-13-277.

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

Nicolas Radomski  1 , Adélaïde Roguet, Françoise S Lucas, Frédéric J Veyrier, Emmanuelle Cambau, Héberte Accrombessi, Régis Moilleron, Marcel A Behr, Laurent Moulin
Affiliations

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

Nicolas Radomski et al. BMC Microbiol. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

PubMed Disclaimer

Figures

Figure 1
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 (CP000479.1), M. paratuberculosis K10 (AE016958.1), M. smegmatis MC2 155 (CP000480.1), M. abscessus ATCC 19977 (CU458896.1), M. gilvum PYG-GCK (CP000656.1), M. vanbaalenii PYR-1 (CP000511.1), Mycobacterium sp. JLS (CP000580.1), Mycobacterium sp. KMS (CP000518.1), Mycobacterium sp. MCS (CP000384.1), and DNA sequences of non-targeted genomes include Corynebacterium aurimucosum ATCC 700975 (CP001601.1), C. diphteriae NCTC 13129 (BX248353.1), C. efficiens YS-314 (BA000035.2), C. glutamicum ATCC 13032 (BX927147.1), C. jeikeium K411 (NC_007164), C. kroppenstedtii DSM 44385 (CP001620.1), C. urealyticum DSM 7109 (AM942444.1), Nocardia farcinica IFM 10152 (AP006618.1), Nocardioides sp. JS614 (CP000509.1), Rhodococcus erythropolis PR4 (AP008957.1), R. jostii RHA1 (CP000431.1) and R. opacus B4 (AP011115.1).
Figure 2
Figure 2
Total (A) and partial representation (B) of the protein number (vertical axe, number in the top of the bars) of Mycobacterium tuberculosis H37Rv genome, according to their similarities with proteins of targeted mycobacterial genomes and proteins of non-targeted genomes (horizontal axes). 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 (CP000479.1), M. paratuberculosis K10 (AE016958.1), M. smegmatis MC2 155 (CP000480.1), M. abscessus ATCC 19977 (CU458896.1), M. gilvum PYG-GCK (CP000656.1), M. vanbaalenii PYR-1 (CP000511.1), Mycobacterium sp. JLS (CP000580.1), Mycobacterium sp. KMS (CP000518.1), Mycobacterium sp. MCS (CP000384.1), and non-targeted genomes include Corynebacterium aurimucosum ATCC 700975 (CP001601.1), C. diphteriae NCTC 13129 (BX248353.1), C. efficiens YS-314 (BA000035.2), C. glutamicum ATCC 13032 (BX927147.1), C. jeikeium K411 (NC_007164), C. kroppenstedtii DSM 44385 (CP001620.1), C. urealyticum DSM 7109 (AM942444.1), Nocardia farcinica IFM 10152 (AP006618.1), Nocardioides sp. JS614 (CP000509.1), Rhodococcus erythropolis PR4 (AP008957.1), R. jostii RHA1 (CP000431.1) and R. opacus B4 (AP011115.1).
Figure 3
Figure 3
Mycobacteria quantification in environmental samples and comparison to reference methods. A) Quantification in drinking water samples (n = 90) was performed by culture method (Method A: Le Dantec et al. 2002) [28], and the new real-time PCR targeting the atpE gene (locus Rv1305 in M. tuberculosis genome) applied to DNA extracted by commercial spin column procedure (Method B: Qiagen kit extraction), or reference DNA extraction procedure (Method C: Radomski et al. 2011) [29]. B) Quantification in lake samples (n = 15) was performed measured by real-time PCR targeting 16S rRNA (Radomski et al. 2010) [17] or atpE genes.
See this image and copyright information in PMC

References

    1. Kazda J. In: The ecology of mycobacteria: Impact on animal’s and human’s health. Kazda J, Pavlik I, Falkinham JO, Hruska K, editor. Vol. 1. Dordrecht Heidelberg London New York: Springer; 2009. The chronology of mycobacteria and the development of mycobacterial ecology; pp. 1–11.
    1. Radomski N, Cambau E, Moulin L, Haenn S, Moilleron R, Lucas FS. Comparison of culture methods for isolation of nontuberculous mycobacteria from surface waters. Appl Environ Microbiol. 2010;76(11):3514–3520. doi: 10.1128/AEM.02659-09. - DOI - PMC - PubMed
    1. Adékambi T, Drancourt M. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol. 2004;54(6):2095–2105. doi: 10.1099/ijs.0.63094-0. - DOI - PubMed
    1. Gomila M, Ramirez A, Lalucat J. Diversity of environmental Mycobacterium isolates from hemodialysis water as shown by a multigene sequencing approach. Appl Environ Microbiol. 2007;73(12):3787–3797. doi: 10.1128/AEM.02934-06. - DOI - PMC - PubMed
    1. Mendum TA, Chilima BZ, Hirsch PR. The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil. FEMS Microbiol Lett. 2000;185(2):189–192. doi: 10.1111/j.1574-6968.2000.tb09060.x. - DOI - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources