title: Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR.
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.
authors.affiliation: Department of Oral Microbiology, Academic Center for Dentistry Amsterdam, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.
authors.forename: Khalil
authors.initials: K
authors.lastname: Boutaga
authors.forename: Arie Jan
authors.initials: AJ
authors.lastname: van Winkelhoff
authors.forename: Christina M J E
authors.initials: CM
authors.lastname: Vandenbroucke-Grauls
authors.forename: Paul H M
authors.initials: PH
authors.lastname: Savelkoul
mesh_terms: D000328:Adult; D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics / Q000302:isolation & purification; D001421:Bacteria, Anaerobic / Q000235:genetics* / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods* / Q000706:statistics & numerical data; D041963:Bacteroidetes / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003773:Dental Plaque / Q000382:microbiology; D005673:Fusobacterium / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D006801:Humans; D010462:Peptostreptococcus / Q000235:genetics / Q000302:isolation & purification; D010518:Periodontitis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D018713:Prevotella intermedia / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S