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KMID : 0362320080330060537
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Journal of Korean Academy of Operative Dentistry
2008 Volume.33 No. 6 p.537 ~ p.544
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SELECTIVE DETECTION OF VIABLE ENTEROCOCCUS FAECALIS USING PROPIDIUM MONOAZIDE IN COMBINATION WITH REAL-TIME PCR
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Kim Sin-Young
Lee Seung-Jong
Kim Eui-Seong
Seo Deog-Gyu
Song Yun-Jung
Jung Il-Young
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Abstract
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Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.
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KEYWORD
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E. faecalis, real-time PCR, propidium monoazide, chlorhexidine
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