Affiliation:
1. Département Eaux et Environnement, Institut Pasteur de Lille, F-59019 Lille Cedex, France
Abstract
ABSTRACT
PCR-based methods have been developed to rapidly screen for
Legionella pneumophila
in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for
L. pneumophila
that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable
L. pneumophila
cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the
L. pneumophila
PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of
L. pneumophila
cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating
L. pneumophila
bacteria in water and, in comparison with conventional qPCR techniques used to monitor
Legionella
, has the advantage of selectively amplifying only viable cells.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
123 articles.
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