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 μg/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.
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Research Article|
September 01 2008
Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment
D. Inoue;
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
E-mail: [email protected]
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H. Tsutsui;
H. Tsutsui
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
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Y. Yamazaki;
Y. Yamazaki
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
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K. Sei;
K. Sei
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
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S. Soda;
S. Soda
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
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M. Fujita;
M. Fujita
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
2Kochi National College of Technology, 200-1 Monobe Otsu, Nankoku, Kochi 783-8508, Japan E-mail: [email protected]
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M. Ike
M. Ike
1Division of Sustainable Energy and Environmental Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]
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Water Sci Technol (2008) 58 (5): 1107–1112.
Citation
D. Inoue, H. Tsutsui, Y. Yamazaki, K. Sei, S. Soda, M. Fujita, M. Ike; Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment. Water Sci Technol 1 September 2008; 58 (5): 1107–1112. doi: https://doi.org/10.2166/wst.2008.474
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