Mutans Streptococci from Clinical Samples Using Real-time Quantitative PCR

Mutans streptococci (MS), e.g., Streptococcus mutans (Sm) and Streptococcus sobrinus (Ss), are associated with dental caries. Furthermore, the Sm to total streptococci (TS) and Ss to Sm ratios have been reported to correlate with caries prevalence. Therefore, detection and quantification of MS can be used to predict caries risk. Objectives: Real-time quantitative polymerase chain reaction (qPCR) was used for detection and quantification of Sm, Ss and TS in oral samples. Methods: Dental plaque, tongue-scrapings and stimulated saliva samples (n=388) were collected from 54 pre-school-aged and 59 school-aged children living in a high caries risk community. Cell lysis and DNA extraction was followed by SYBR Green based qPCR using primers specific for Sm, Ss and TS. UA 159 genomic DNA was used to generate a standard curve from which the “copy number” (CN/ml) of Sm, Ss and TS was extrapolated from quantification cycle values.  Sm UA 159 and Ss 6715 were used as positive controls. Results: The detection limit of this qPCR was determined to be 70 CN/ml. Sm was detected from 96.6% of samples (375/388) while Ss was only found in 2.3 % of samples (9/388). Mean qPCR CN/ml of samples were: Sm (1.03x10⁷), Ss (2.76x10⁵) and TS (2.97x10⁹) CN/ml.  Mean qPCR CN/ml were found to be more sensitive than standard plate counting for Sm and TS. Of the children with detectable Sm, the mean ratio of Sm/TS was 0.027; and of the 8 children with detectable Ss, the mean ratio of Ss/Sm was 0.25.  Interestingly, Ss was not found to be associated with dental caries activity (2/8 children); and Sm was not detected in 4 children with a history of caries.  Conclusion: Most children in this study have detectable levels of Sm by qPCR whereas most do not have detectable levels of Ss.