Objectives: A real-time PCR based on SYBR-Green I fluorescence was developed for quantifying multi-species carigenic strains in dental plaque, and assessing the relationship between the level of these cariogenic pathogens and dmft index. Methods: Species-specific primers for Streptococcus mutans ,Streptococcus sobrinus,Actinomyces viscosus, Actinomyces naeslundii, and universal primer for a broad range of bacteria were designed, respectively. Conventional PCR assays used to confirm the specificity and universality of the primers. Consequently, for each real-time PCR, standard curves were plotted for each primer using the Ct values obtained by amplifying successive 10-fold dilutions of a known concentration of DNA. Dental plaque were obtained from forty-five Chinese pre-school child subjects, 15 in caries-free group (dmft=0), 15 in moderate-caries group (dmft=4-6) and 15 in high-caries group (dmft >8), ranging in age from 3 to 5 years old. After genomic DNA were isolated from these specimen, the amount of the targeted microorganisms and the total number of bacterial cells were determined by real-time PCR. Calculations were performed using the SAS 6.12 Software. Results: The percentages of these microorganisms in the three group were 2.65±0.9, 8.84±5.99, 13.14±6.87 for S.mutans, 1.34±0.53, 4.27±3.18, 5.99±3.75 for S.sobrinus, 3.15±3.39, 7.02±4.5, 9.82±6.6 for A.viscosus and 1.63±0.79, 2.70±1.49, 4.03±2.04 for A.naeslundii. ANOVA show statistically significant difference between the level of S.mutans, A.viscosus and S.sobrinus, A.naeslundii in each groups (p<0.01).The percentages of the 4 targeted bacteria in high-caries group were higher than those in caries-free group (p<0.01). Conclusion: The application of Real-time PCR proved to be a highly reliable, specific and time-saving approach for identification and enumeration carious-causing bacteria, which are essential for an accurate evaluation of caries susceptibility and the risk of developing caries.
This work was supported by a grant from National Science Foundation of China (No. 30572039), and the Project of the "Tenth Five-Year-Plan" of China (No.2004BA720A24)