Rapid Multiplex Quantitation of Oral Pathogens Using Novel Q-PCR Technology

The tendency of the standard plate count (SPC) to underestimate streptococci in oral samples has been recognized for years. This is partially due to uncontrollable aspects of sample collection, transport and viability. Count inaccuracies are further skewed because the inherent chain-like growth of the streptococci does not effectively adhere to the theoretical assumption that one organism produces one colony on an agar plate. Purpose: The purpose of this study is to investigate the use of a novel multiplex quantitative polymerase chain reaction (Q-PCR) to introduce speed, accuracy, reproducibility and cost reduction, while simultaneously quantitating multiple cariogenic organisms in stimulated saliva samples to resolve the traditional problems of quantitating organisms in oral samples. Methods: Five microliters of ten-fold serially diluted S. mutans, S. sobrinus, and S. sanguinis DNA was used as template in separate 25 µl Plexor Q-PCR reactions that were thermalcycled and subjected to melting curve analysis with a BioRad IQ5 gradient thermalcycler. Cycle threshold (Ct) values versus copy number standard curves were constructed for each organism. Likewise, a total streptococci standard curve was constructed using 2 µl of each diluted complex mixture containing equal amounts of streptococcal genomic DNA isolated from a variety of oral streptococci. Pure broth cultures of known cell densities of S. mutans, S. sobrinus, and S. sanguinis as well as stimulated saliva samples were subjected to standard plating on Gold's and MS agar as well as Q-PCR. Data from the two methods were statistically evaluated using a paired t-test. Results: The multiplex quantitation method, gave counts that were higher and more reproducible, compared to the SPC. Conclusion: The improvement in speed, reproducibility, accuracy and reduction of workload and expense make Plexor multiplex Q-PCR assays an attractive alternative for quantitative assessment of saliva samples, especially when high throughput is needed.

Supported by NIDCR grants: DE016684 and T32 DE017607