Live/dead real-time polymerase chain reaction to assess new therapies against dental plaque-related pathologies

DNA-based methodology for the identification and detection of specific bacteria in dental plaque offers advantages over culturing techniques. One drawback of current molecular techniques like real-time quantitative polymerase chain reaction (RT-QPCR) is that they are not able to distinguish between live or dead bacteria. To overcome this problem an assay was assessed to discriminate between viable or dead bacteria using DNA intercalating substances, propidium monoazide (PMA) and ethidium monoazide (EMA) in combination with RT-QPCR. The assay was tested on oral pathogens: Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. To determine the effectiveness of EMA and PMA, different concentrations (from 5 to 100 mu g ml(-1)) of the substances were added to viable or heat-killed suspensions of both organisms (ranging from 10(8) to 10(4) colony-forming units ml(-1)). Afterwards, PMA was tested on mixtures of varying ratios of viable and dead cells. After DNA extraction, RT-QPCR was performed using species-specific primers. Both compounds inhibited PCR amplification from dead cells. The EMA treatment resulted in the largest signal decrease but EMA also inhibited DNA amplification from viable cells. For this reason, PMA was selected for use in further experiments. It was shown to be efficient in allowing selective PCR detection of only viable cells in mixtures containing both viable and dead cells. The amount of amplified DNA corresponded to the percentage of viable cells in the sample. The developed assay will potentially be useful for assessing bacterial loads remaining after disinfection protocols without interference by nonviable bacteria.