A novel closed-tube quantitative--PCR method for enumerating Porphyromonas gingivitis, Prevotella intermedia and Actinobacillus actinomycetemcomitans.

Enumeration of specific periodontopathogens in subgingival plaque samples has been problematic because of either lack of sensitivity, specificity or the time taken to identify the organisms. These problems can be overcome using PCR, but quantification by this technique is more difficult. We report a simple quantitative PCR method developed for enumerating Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in clinical samples. The method relies upon inclusion of ethidium bromide in a closed-tube PCR reaction and measurement of resultant amplicons by quantifying the fluorescence generated when UV-light is passed through the walls of the amplification tube. Hence, both PCR and detection are performed in the same tube. The technique was compared with a quantitative competitive PCR and a commercial colorimetric quantitative PCR and proved to be at least as sensitive, specific and reliable for enumeration of the target bacteria. However, its speed and convenience make it particularly useful for large-scale analyses in both clinical laboratories and epidemiological studies.