• Journal of Korean Society of Water and Wastewater
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• v.23 no.2
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• pp.199-205
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• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

• Journal of fish pathology
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• v.31 no.2
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• pp.93-99
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• 2018

Edwardsiella tarda predominantly causes edwardsiellosis in fish at high temperature, but is rarely isolated from water when water temperature is low. However, E. tarda is viable but nonculturable (VBNC) in low water temperature, but it can be revived when water temperature rises and cause disease to fish. Therefore, in order to prevent disease, it is very important to identify pathogens that are in the VBNC state in environmental water. In this study, E. tarda cells in the VBNC state were detected by the ethidium monoazide (EMA)-PCR method using the low-temperature oligotrophic sea water microcosm obtained by inoculation of E. tarda at a concentration of $10^8CFU/ml$. In order to distinguish between live and dead bacteria in E. tarda, each sample was treated with EMA at different concentrations, photoactivated with a 500 W halogen lamp, and PCR was performed with E. tarda specific primer. At the concentration of $10^7CFU/ml$ bacterium, DNA amplification was observed only in the live cells when treated with $60{\mu}g/ml$ of EMA, and smaller amounts of live cells could be distinguished from dead cells by adjusting the EMA concentration. In addition, the VBNC cells of E. tarda in the oligotrophic low temperature seawater microcosm were estimated to be in the range of $10^4{\sim}10^5CFU/ml$ by EMA-PCR. Therefore, it is possible to detect VBNC cells that will act as potential pathogens in environmental water using EMA-PCR method, and quantitative confirmation using concentration change is also possible.

• Food Science and Biotechnology
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• v.18 no.3
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• pp.788-792
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• 2009

Gamma $({\gamma})-irradiation$ can be used to control pathogens such as Vibrio vulnificus in seafood. The effects of irradiation on microbial cell populations (%) have been studied in order to develop detection methods for irradiated foods. The method used in this study was ethidium bromide monoazide (EMA) real-time polymerase chain reaction (PCR), using V. vulnificus specific primer, EMA, and $SYBR^{(R)}$ Green to discriminate between ${\gamma}-irradiated$ and non-irradiated cells. Confocal microscope examination showed that ${\gamma}-irradiation$ damaged portions of the cell membrane, allowing EMA to penetrate cells of irradidated V. vulnificus. ${\gamma}-Irradiation$ at 1.08 KGy resulted in log reduction ($-1.15{\pm}0.13$ log reduction) in genomic targets derived from EMA real-time PCR. The combination cold/heat shock resulted in the highest ($-1.74{\pm}0.1$ log reduction) discrimination of dead irradiated V. vulnificus by EMA real-time PCR.