Quantification of bacteria in oral samples by competitive polymerase chainreaction

Information about the total amount of bacteria in oral samples contributes to assessment of an individual's risk of contracting dental caries or devel oping periodontitis and the prediction of that individual's clinical course . Since existing techniques are often time-consuming and expensive, it seem ed attractive to look for alternative methods for the quantification of eub acteria. With their high specificity and sensitivity, polymerase chain-reac tion (PCR) techniques have the potential of supplying fast and reliable res ults. We developed a method of competitive PCR for the quantification of eu bacteria. We designed forward and reverse PCR primers which bind to highly conserved sequences of the bacterial 16S rRNA gene. A homologous competitor was synthesized with Escherichia coli 16S rDNA as a template, with the rev erse primer and a hybrid primer which binds 67 bases downstream to the forw ard primer and carries the forward primer sequence at its 5' end. Specifici ty controls with 30 different bacterial species, 5 archaea, 3 fungi, human astrocytoma cells, and rat hepatoblastoma cells were carried out. Results w ere positive for all eubacteria and negative for all other cells tested. Ca libration curves were obtained by co-amplification of known amounts of E. c oli cells in the presence of the homologous competitor. The developed metho d was successfully applied to assessment of the accumulation of bacteria du ring an oral hygiene cessation experiment, The competitive PCR method prove d to be a reliable and fast method for the quantification of bacterial DNA and cultured eubacteria, as well. as of bacteria in biological samples. It may find further applications not only in periodontology and cariology but also in other fields of medical microbiology.