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Ethidium monoazide-PCR for the detection of viable Escherichia coli in aquatic environments  

Lee, Gyucheol (한국수자원공사)
Kim, Hyunjeong (한국수자원공사)
Lee, Byunggi (한국수자원공사)
Kwon, Soonbok (한국수자원공사)
Kim, Gidon (한국수자원공사)
Lee, Sangtae (한국수자원공사)
Lee, Chanhee (충북대학교 생명과학부 미생물학과)
Publication Information
Journal of Korean Society of Water and Wastewater / v.23, no.2, 2009 , pp. 199-205 More about this Journal
Abstract
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.
Keywords
ethidium monoazide; polymerase chain reaction; real-time polymerase chain reaction;
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