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

Abstract

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.

Keywords: multiplex; periodontal pathogens; periodontitis; real‐time PCR.

Figure 1

SYBR Green fluorescence during real‐time amplification with species specific oligos: (a) A gradual delay in amplification and an increase in C _q are apparent, which is consistent with 10‐fold gradual decrease in template concentration. (b) The standard curve representing the dilution series indicated in “A”. The C _q signal is titrated from 0.5 fg to 50 pg and shown in the standard curve. The slope indicates high level of polymerase chain reaction efficiency

Figure 2

Real‐time amplification demonstrating oligo specificities: SYBR Green fluorescence during real‐time amplification with species‐specific TaqMan oligos and genomic DNA (black lines), bacteria‐specific TaqMan oligos with a mixture of bacterial genomic DNA as template (green lines), and mixed TaqMan oligos and individual genomic DNA as template (red lines). The amount of each oligo and each genomic DNA concentrations was identical whether included individually or in mixture

Figure 3

Comparison of multiplex and singleplex assays with TaqMan probe‐based detection. (a) multiplex amplification with mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA as template. (b–f) Singleplex TaqMan amplification signals when species‐specific TaqMan oligos, species‐specific TaqMan probes, and specific target DNA were used (lines with markers) relative to multiplex amplification signal when mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA were used (straight lines)