Real-time PCR for detection of Glucosyl Transferase in clinical samples

Methods: Saliva samples and pure cultures of Streptococcus oralis, Streptococcus sanguis biovars, Streptococcus mitis biovars, Streptococcus gordonii and Lactobacillus species were lysed by using an optimized MagnaPure^® protocol, enabling a maximal pcr signal with specific GTF primers and probes. Positive signals from bacterial species other than Streptococcus mutans were cloned and sequenced to determine whether these DNA sequences are homologous to S. mutans GTF.

Results: Correlation analyses of the number of S. mutans cells in pure culture and the TaqMan Ct-values revealed a correlation coefficient of r = 0.999. It was found that S. sanguis biovar 3 also gave a positive pcr-signal. The Ct-values of S. sanguis were higher, corresponding to a 10⁵ lower DNA concentration than the Ct-value of S. mutans. The resulting PCR-product of S. sanguis was cloned, sequenced and proved to be a part of a GTF gene closely related to that of S. mutans.

Conclusion: By using real-time PCR, we have shown that GTF-genes can be detected from S. mutans and that similar genes fragments are also present in S. sanguis. It is not known whether the genes of S. sanguis will result in an active GTF enzyme. This method enables to study the adhesive capacity of dental plaque in relation to caries activity.