Endpoint quantitative PCR assays for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans

Background: Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. Objective: Our goal was to develop qPCR assays which would allow endpoint quantification. Materials and methods: 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the Amplifluor™ qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. Results: Optimized standard curves were linear in log-log or semilog fits over a range of 100-10⁶ cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. Conclusions: Amplifluor™ qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories.