title: Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment.
abstract: Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.
authors.affiliation: Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. inoue@wb.see.eng.osaka-u.ac.jp
authors.forename: D
authors.initials: D
authors.lastname: Inoue
authors.forename: H
authors.initials: H
authors.lastname: Tsutsui
authors.forename: Y
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: K
authors.initials: K
authors.lastname: Sei
authors.forename: S
authors.initials: S
authors.lastname: Soda
authors.forename: M
authors.initials: M
authors.lastname: Fujita
authors.forename: M
authors.initials: M
authors.lastname: Ike
mesh_terms: D001386:Azides / Q000737:chemistry*; D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D050296:Microbial Viability / Q000235:genetics*; D008962:Models, Theoretical; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium