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

Radomski N, Roguet A, Lucas FS, Veyrier FJ, Cambau E, Accrombessi H, Moilleron R, Behr MA, Moulin L - BMC Microbiol. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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. nicolas.radomski.phd@gmail.com.

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.

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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.
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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.

Mentions: In order to compare with culture-based method (Method A) [28], and evaluate the impact of extraction methods on the quantification process by the new real-time PCR, we used two DNA extraction procedures (Method B and C) on water distribution samples: a commercial kit (Method B) and a published phenol-chloroform extraction (Method C) [29]. DNA extraction from tap water significantly influenced the result of mycobacteria detection by atpE real-time PCR (Figure 3A). Detection levels from DNA extracted by the kit (Method B) were significantly higher (Wilcoxon signed-rank test, n = 90, p = 0.002) than those from DNA extracted by phenol/chloroform procedure (Method C). The percentage of positive samples was significantly higher (Chi-square test, n = 180, df = 1, p = 0.021) when performing the real-time PCR with the DNA extracted by method B (33/90), compared to method C (19/90). In order to evaluate the new real-time PCR method, we compared the levels of mycobacteria detected in water distribution samples with a published culture method called method A [28]. Using the method A, Mycobacterium spp. colonies were obtained from 76% of tap water samples.


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

Radomski N, Roguet A, Lucas FS, Veyrier FJ, Cambau E, Accrombessi H, Moilleron R, Behr MA, Moulin L - BMC Microbiol. (2013)

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.
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Related In: Results  -  Collection

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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.
Mentions: In order to compare with culture-based method (Method A) [28], and evaluate the impact of extraction methods on the quantification process by the new real-time PCR, we used two DNA extraction procedures (Method B and C) on water distribution samples: a commercial kit (Method B) and a published phenol-chloroform extraction (Method C) [29]. DNA extraction from tap water significantly influenced the result of mycobacteria detection by atpE real-time PCR (Figure 3A). Detection levels from DNA extracted by the kit (Method B) were significantly higher (Wilcoxon signed-rank test, n = 90, p = 0.002) than those from DNA extracted by phenol/chloroform procedure (Method C). The percentage of positive samples was significantly higher (Chi-square test, n = 180, df = 1, p = 0.021) when performing the real-time PCR with the DNA extracted by method B (33/90), compared to method C (19/90). In order to evaluate the new real-time PCR method, we compared the levels of mycobacteria detected in water distribution samples with a published culture method called method A [28]. Using the method A, Mycobacterium spp. colonies were obtained from 76% of tap water samples.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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. nicolas.radomski.phd@gmail.com.

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.

Show MeSH
Related in: MedlinePlus