Multiplex real-time PCR detection and relative quantification of periodontal pathogens

Periodontitis is a chronic inflammatory disease, which is strongly associated with certain pathogenic bacteria. The aim of this study was to develop a real-time multiplex polymerase chain reaction (PCR) assay to detect and quantify bacterial species associated with periodontitis. We targeted detection and relative quantification of the following five bacterial species relevant to periodontal diseases: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. The conserved regions of the genome of these species were targeted with oligos and TaqMan probes in real-time PCR assays. The species-specific TaqMan oligos and TaqMan probes showed no cross-amplification, and there was no loss of amplification yield in multiplex real-time PCR assays. All five bacterial targets were amplified analogous to the template concentrations used in these assays. This multiplex real-time PCR strategy could potentially be used to detect the bacterial species in periodontal pockets of patients with periodontal diseases. This assay may also serve as a quick tool for profiling and quantifying bacteria relevant to periodontal diseases and likely be a valuable tool for clinical translational research.