Abstract

Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with quantitative anaerobic culture for detection and quantification of 5 prominent periodontal pathogens. Real-time PCR assays with the 16s rRNA genes of Actinobacillus actinomycetemcomitans, Prevotella intermedia, Tannerella forsythensis, Peptostreptococcus micros and Fusobacterium spp. were developed. The PCR was validated on pure cultures of various bacterial strains. Subsequently, subgingival plaque samples from 259 adult patients with periodontitis were analyzed with quantitative anaerobic culture and real-time PCR. A standard curve for DNA quantification was created for each primer-probe set based on colony-forming units equivalents.

All bacterial species were correctly identified. The lower limits of detection by PCR varied between 1–50 colony-forming units equivalents depending on the species. No cross-reactivities with heterologous DNA of other bacterial species were observed. Real-time PCR results showed a high degree of agreement with anaerobic culture results. Real-time PCR is a reliable alternative for diagnostic quantitative anaerobic culture of subgingival plaque samples.

Keywords: Periodontitis; Subgingival plaque; Quantitative real-time PCR; Anaerobic culture

Article Outline

1. Introduction

2. Materials and methods

2.1. Study population and sample collection

2.2. Microbiological procedures

2.2.1. Identification of anaerobic isolates

2.2.2. Growth of reference strains

2.3. DNA isolation from plaque samples and bacterial reference cultures

2.4. Validation of the real-time PCR

2.4.1. Design of PCR primers and probes

2.4.2. Validation of the primer/probe sets for real-time PCR

2.4.3. Inhibition

2.5. Genome sequence analysis

2.6. Statistics

3. Results

3.1. Sensitivity and specificity of TaqMan assay

3.2. Reproducibility and reliability

3.3. DNA isolation and PCR inhibition controls

3.4. Real-time PCR versus anaerobic culture

4. Discussion

Acknowledgements

References