Streptococcus mutans Counts from Clinical Samples Using Quantitative Real-time PCR

Streptococcus mutans colonization of the oral cavity is closely associated with dental caries, therefore, its quantitaton is considered as an important indicator of caries risk. Objectives: This study used SYBR GREEN based real-time PCR to quantify S. mutans in oral samples for comparison to results obtained by traditional standard plate counts (SPC). Methods: Saliva, plaque and tongue-scrape samples, were collected. SPC were performed using a Spiralplater to plate a ten-fold dilution series ranging from 10^-1- 10^-4 on duplicate and alternating pairs of Gold's and MS agar. The plates were incubated anaerobically at 37ºC for 48 hours. Colonies were counted and the CFU/ml was calculated. DNA extraction was performed on each clinical sample and served as a template for real-time PCR using S. mutans specific primers. A ten-fold dilution of a known concentration of UA 159 (determined by flow cytometry) was prepared and a standard curve was constructed to estimate samples cfu/ml from Ct values. Results: Comparison of averages as well as sample to sample comparisons, in most cases, revealed that S. mutans counts obtained by real-time PCR were higher than those obtained by SPC. Furthermore, the probability of detecting S. mutans from clinical samples was higher for real-time PCR than SPC. Conclusion: Higher cell counts by real-time PCR could be due to many factors such as cell viability, and growth in aggregates which is characteristic of S. mutans. Real-time PCR has the potential to become a preferred methodology for quantitation in oral samples because of its accuracy, sensitivity and efficiency. These results suggest that real-time PCR should be further explored as a means of quantifying S. mutans and other bacteria in oral samples.

This study is supported by NIDCR grant #DE016684