Polymerase chain reaction for the identification of Porphyromonas gingivalis collagenase genes

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Abstract
Porphyromonas gingivalis has been shown to exhibit genetic diversity possibly resulting in variation of virulence. In the present study a potential virulence factor was targeted for the detection of P. gingivalis. A 548 bp fragment of the collagenase gene (prtC) from Porphyromonas gingivalis ATCC 33277 was amplified by polymerase chain reaction (PCR) using oligonucleotides derived from the middle portion of prtC. From 16 of 21 clinical P. gingivalis strains, a PCR product of similar size to the prtC could be obtained. These 16 P. gingivalis strains were confirmed as positive for prtC using DNA hybridization with a digoxigenin-labeled prtC PCR product as a probe. In 12 of the 16 prtC positive strains, the restriction analysis of the PCR products revealed fragment patterns identical to the known sequence. In the other 4 prtC positive strains, 4 distinct patterns were found. Of these strains, nucleotide sequence analysis of a 400 bp PCR product stretch revealed 79.1%, 83.0%, 84.8 and 89.5% homology with the known nucleotide sequence for this specific region. Sequence analysis of the PCR products from the ATCC 33277 strain demonstrated 93.7% homology. The limit of detection for the PCR was about 100 organisms. None of the other 48 tested strains of 16 bacterial species derived from oral and extraoral infections yielded a PCR product. The PCR was also used for the detection of prtC sequences in dental plaque. Our data indicate that not all P. gingivalis strains have prtC. Nucleotide heterogeneity exists among P. gingivalis with prtC. Using a potential virulence factor for the detection of putative periodontal pathogens such as P. gingivalis may be valuable for the epidemiology of infection and clinical diagnosis of periodontal diseases.

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