In Vivo Quantitative Evaluation of Live and Dead Bacteria in Root Canal Infection by Using Propidium Monoazide with Real-Time PCR

Abstract

INTRODUCTION: For selective detection of viable bacteria with molecular methods, propidium monoazide (PMA) treatment has been successfully applied to a wide range of bacteria. The purpose of this study was to compare the quantity of live cells with the total amounts of both live and dead cells before and after chemomechanical preparation by using PMA in combination with real-time polymerase chain reaction (qPCR). METHODS: Twenty-one teeth with pulp necrosis and a periapical lesion were included. Bacterial sampling of the root canals was performed before (S1) and after (S2) chemomechanical root canal treatment. Each sample was separated into 2 different tubes. PMA was added to one of the tubes, and the other was left untreated. Then, DNA extraction and qPCR were performed. To evaluate the validity of the PMA treatment, the defined mixtures containing different ratios of live and dead cell suspensions of Enterococcus faecalis were either subjected to PMA treatment or not subjected to PMA treatment, followed by qPCR quantification. RESULTS: A paired t test showed a highly significant difference in the mean threshold cycle values between S1 with and without PMA (P = .0002), and this difference (0.89) was similar to that (0.96) obtained from the samples consisting of 80% live cell suspension and 20% dead cell suspension of E. faecalis. The threshold cycle values between the S2 samples with and without PMA were also significantly different (P = .0134), and this difference (0.37) was similar to that obtained from the 100% live cell suspension of E. faecalis (0.42). CONCLUSIONS: PMA in conjunction with qPCR appeared to be useful in analyzing the primary infections of root canals because there were significant amounts of dead bacteria in the root canals. Although the use of PMA treatment in post-preparation samples significantly reduced the detection of dead bacteria, this difference was still small, so further studies should be carried out to confirm the necessity of PMA treatment.