Enumeration of viable cariogenic pathogens assessed by PMA-based real-time PCR

Objectives: Streptococcus mutans and S. sobrinus are associated with the development of dental caries in humans. Various methods have been developed to identify S. mutans and/or S. sobrinus. Most reported diagnosis systems are unsuitable for monitoring the viable cell number and/or ratio of live/dead cariogenic bacteria in oral specimens. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and cross-link the DNA via photoactivation, thereby inhibiting amplification. In this study, we developed an assay for selective analysis of viable S. mutans and S. sobrinus by using PMA in combination with real-time PCR. Methods: Initially, real-time PCR primers were designed by S. mutans- and S. sobrinus-specific regions. To determine the effectiveness of PMA, different concentrations (2.5–250 µM) of the substance were added to viable suspensions. Afterward, PMA was tested on mixtures of varying ratios of viable and autoclave-killed cells. After DNA extraction, real-time PCR analysis was performed. Results: PMA treatment effectively prevented DNA amplification from dead cells. Of the various concentrations, 25 µM PMA demonstrated the strongest inhibition of dead cell DNA amplification. Using this concentration, various ratios of live/dead cells were examined, and no inhibition by dead cells was observed. Conclusions: This assay will potentially be useful for assessing cariogenic pathogens without interference by nonviable bacteria. This work was supported by a Grant-in-Aid (C) 22592341 (to A.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.