Evaluation of bacterial viability in root canal biofilms using propidium monoazide treatment with real-time PCR
Dept. of Dentistry/박사
The goal of endodontic treatment is the elimination of root canal bacteria which are the cause of apical periodontitis. Polymerized chain reaction (PCR) can detect bacteria more rapidly than conventional culturing method. However, PCR method 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. It is used before DNA extraction, resulting in selective DNA detection of viable bacteria. PMA penetrates cells with compromised membranes and intercalates with dead cell DNA upon light exposure, then inhibits PCR amplification. The purpose of this study was to investigate the effectiveness of PMA in conjunction with real-time PCR for evaluation of bacterial viability in root canal biofilms.Firstly, to evaluate the relationship between the proportion of viable bacteria and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead Enterococcus faecalis were used. Secondly, to confirm the effect of universal primer on root canal bacteria, real-time PCR of 4 different kinds of bacteria were performed using the universal primer. In clinical study, 21 teeth with pulp necrosis and periapical lesion were included. Bacterial sampling in the root canals was performed using paper points before and after chemomechanical root canal treatment. Each sample was separated to 2 different tubes, 1.25 μl of PMA was added to one of them and the other one was left untreated. After that, DNA extraction and real-time PCR were performed. Ct values were automatically calculated by the real-time PCR software system and the higher the Ct values, the lower the amount of bacteria. As a result, Ct differences between without PMA and with PMA treatment decreased proportionally with increasing viable E. faecalis percentage. And, 4 different
kinds of bacteria responded well to the real-time PCR reaction using universal primer. In clinical study, paired t-test showed a highly significant difference in Ct values between S1 without PMA and S1 with PMA (p=0.0002) and it also showed significant difference in Ct values between S2 without PMA and S2 with PMA (p=0.0134). A significant differences were found in Ct values between S1 without PMA and S2 without PMA (p=0.003) and Ct values between S1 with PMA and S2 with PMA (p=0.0238). Live and dead root canal bacteria were totally detected without PMA pretreatment, and only live bacteria were selectively detected with PMA pretreatment. It means that some amount of dead bacteria were present before and after chemomechanical root canal treatment. Total bacterial volume decreased after chemomechanical treatment and the amount of viable bacteria also decreased. In conclusion, PMA pretreatment with real-time PCR method is effective for selective detection of viable root canal bacteria.