An mRNA-based real-time PCR viability assay for oral bacteria

DNA-based methods for the detection of oral bacteria are unable to determine the viability of those species. Because oral bacteria are most often found in biofilm communities, the DNA of dead bacteria can be retained within the biofilm architecture for long periods of time following killing. Other methods, such as fluorescence-based viability assays (Live Dead kit, Molecular Probes), can detect whether or not organisms have compromised membranes, but don't directly detect specific species. Objective: The objective of this study was to develop a method using reverse transcription real-time PCR to quantify the viable organisms of a specific species of oral bacteria present within in a complex community. Methods: mRNA has a relatively short half life and therefore is indicative of recently active bacteria. We have developed species-specific primers to the elongation factor tuf. This gene is not significantly regulated by growth phase, media or environmental conditions, thereby minimizing spurious effects on detected numbers of bacteria. Results: Using Aggregatibacter actinomycetemcomitans as our test organism, we were able to detect viability differences in mixed populations of live and EtOH killed bacteria when as few as 20% of the organisms present were viable. Additionally, we were able to reliably identify the presence of A. actinomycetemcomitans in mixed species populations containing up to six different species of bacteria. Calculated bacterial concentrations correlated closely to values estimated based on OD₆₁₀ for the same cultures (r= 0.96, <1% difference). Using this standard curve, the concentration of viable bacteria in a sample were calculated within <20% of the actual value based on OD₆₁₀, which was not statistically different. Conclusion: This assay represents a quantitative means of monitoring the viability of specific organisms in mixed species environments.