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Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR

Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland, kati.hyvarinen{at}helsinki.fi

Saara Laitinen

Finnish Red Cross Blood Service, Helsinki, Finland

Susanna Paju

Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland

Anne Hakala

Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland

Liisa Suominen-Taipale

National Institute for Health and Welfare, Kuopio, Finland

Mikael Skurnik

Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland

Eija Könönen

National Institute for Health and Welfare, Helsinki, Finland, Institute of Dentistry, University of Turku, Turku, Finland

Pirkko J. Pussinen

Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland

Periodontitis is a common chronic multibacterial infection in the tooth-supporting tissues. It has been shown that periodontitis patients carry higher number of disease-associated bacteria than healthy ones. The aim of this study was to generate a novel, single copy gene-based quantitative real-time PCR (qPCR) assay for five major periodontal pathogens — Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, and Tannerella forsythia. The primer/probe sets were designed for conservative lipopolysaccharide-coding gene regions. They proved to be sensitive and able to detect strains representing different serotypes of the target bacteria. The specificity of designed primers was tested using 49 selected bacterial species and no false positive or negative results were observed. We validated the assay with a case-control population, including 165 saliva samples, and proved the diagnostic accuracy by Receiver Operating Characteristic (ROC) curves. All quantified pathogens alone were able to distinguish significantly between the subjects with and without periodontitis, and provided areas under the ROC curve larger than 0.5. The total pathogen burden comprising all five species associated with periodontitis with an area of 0.821 (95% CI, 0.758—0.885, P50.001). Our prominently sensitive and specific assay may have major importance in the diagnosis, prevention, and treatment of periodontitis.

Key Words: dentistry • infection • LPS • oral pathogen burden • saliva

This version was published on August 1, 2009

Innate Immunity, Vol. 15, No. 4, 195-204 (2009)
DOI: 10.1177/1753425908101920


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