Selective detection of viableEnterococcus faecalisusing propidium monoazide in combination with real-time PCR

Abstract

ABSTRACT Propidium monoazide와real-time PCR을이용한살아있는Enterococcus faecalis의선택적인검출 김신영∙이승종∙김의성∙서덕규∙송윤정∙정일영*연세대학교치과대학치과보존학교실 SELECTIVE DETECTION OF VIABLE ENTEROCOCCUS FAECALISUSING PROPIDIUM MONOAZIDE IN COMBINATION WITH REAL-TIME PCR Sinyoung Kim, Seungjong Lee, Euiseong Kim, Deoggyu Seo, Yoonjung Song, Ilyoung Jung* Department of Conservative Dentistry, College of Dentistry, Yonsei University 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 DNAafter cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced.The purpose of this study is to evaluate the applicability of t he PMA treatment and real-time PCR methodfor cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX onE. faecalisusing PMA treatment in combination with real-time PCR.Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signalsafter PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relativedifference of viable cells using PMA treatment in combination with real-time PCR was compared with CFUby plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecaliswas measured using PMA treat-ment 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 CFUby plate counting until Optical density (OD) value remains under 1.0. However, viable cells measuredby real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing.3.Relative difference of viable E. faecalisdecreased more after longer application of 2% CHX.