Streptococcus mutans and
Streptococcus sobrinus are considered the primary aetiologic agents of human dental caries and thus most anti-cariogenic preventive strategies aim to suppress these acid-producing bacteria.
Objective: The aim of this project was to develop a PCR method for the detection and quantification of
S. mutans and
S. sobrinus in human saliva samples suitable for use in determining the efficacy of anti-mutans regimes.
Methods: Chromosomal DNA was extracted from bacteria by treating saliva samples with recombinant Zoocin A for 30 minutes, followed by boiling in lysis solution (Triton X-100/EDTA) for 10 minutes. To remove salivary components that may interfere with the PCR, the released DNA was precipitated with ethanol and redissolved in water. Oligonucleotides specific to the glucosyltransferase genes (
gtf B of
S. mutans and
gtf I of
S. sobrinus [Oho et al. Oral Microbiol Imuunol.15:258-262, 2000]) were used to prime the PCR using the extracted DNA as template.
Results: The specificity of the primers was verified by testing against catalogued isolates of
S. mutans,
S. sobrinus and a variety of other streptococcal species. This method consistently detected 100 bacteria in 10 µl of saliva. The detection level was not influenced by growth of bacteria in sucrose, which resulted in copious production of extracelluar polysaccharide that could have interfered with DNA extraction and/or amplification.
Conclusion: The described PCR method allowed convenient quantitative detection and differentiation of
S. mutans and
S. sobrinus in saliva samples, and will have application in caries assessment and monitoring programmes. L. S-Y. Park was supported by a University of Otago Research Theme Summer Studentship.