Quantification of Propidium Monoazide (PMA)-treated Endodontic Bacteria with Real-time PCR

Objective: Root canals should be sterilized before root canal obturation. Anaerobiotic culture systems are generally accepted as a method to analyze the sterilization status of a root canal, but it takes one or two weeks to judge. Propidium Monoazide (PMA), fluorescent stain, selectively penetrates into dead bacterial cells, but not into viable bacteria cells, resulting in selective PCR detection of only viable bacteria cells. The aim of this study was to examine correlation with a Ct value of real-time PCR using PMA and conventional method such as CFU count method in vitro, to establish rapid and highly sensitive detection method of bacteria in the root canal. Methods: Viable and dead cell suspensions of Enterococcus faecalis were mixed in defined rations, and examined by following methods; 1) CFU count, 2) LIVE/DEAD^® BacLight^TM Bacterial Viability Kit, and 3) real-time PCR using PMA. The correlation of each value from each method were analyzed. Furthermore, E. faecalis was exposed to 3% EDTA solution and 2.5% sodium hypochlorite (NaOCl) solution. Results: Ct values of samples from real-time PCR with PMA showed distinct differences among samples, and showed high correlation with results of CFU count. On the other hand, the staining with LIVE/DEAD^® BacLight^TM Bacterial Viability Kit was effective method to show sterilization status in a short time, but the number of bacteria quantification was difficult. Conclusion: Combination of PMA staining with real-time PCR method may be effective to quantify bacteria in the root canal in a short time.