Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3' ends with respect to the corresponding regions of the 16S rRNA gene of P. nigrescens, its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease RsaI, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed