Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method

Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomy cetemcomitans are believed to play an important role in adult periodontitis , but the significance of their relative numbers and progress of the diseas e is still unclear. Traditional quantitative methods are generally time-con suming and inaccurate, The aim of this study was to develop a sensitive, qu antitative PCR technique that would be useful for enumerating P. gingivalis , Pr. intermedia and A, actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes wer e employed: the fimbrial gene (fimA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitan s. Competitive templates were constructed either by sequence deletion betwe en primer binding sites or by annealing of the primer binding sites to an a ppropriate DNA core so as to yield products of a different size from that o btained with the target template. Coamplification of target and competitive templates yielded products of expected size and non-specific recognition b y the primers was not found. The sensitivity of the designed primers was 10 0 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. ac tinomycetemcomitans. The three species were found in subgingival plaque sam ples collected from both healthy and diseased sites by the quantitative-com petitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopa thogens, the total number of bacteria in the samples was enumerated by quan titative-PCR with 16S rRNA universal primers (27f and 342r). The findings i ndicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.