Prevalence of four Porphyromonas gingivalis rag alleles among periodontitis patients

Objectives: The rag locus of Porphyromonas gingivalis encodes an immunodominant outer membrane complex predicted to bind an extracellular ligand, and having a role in virulence in an animal model. We have previously identified four divergent alleles of the locus among our laboratory collection of isolates. To investigate the prevalence of the different rag alleles in periodontal disease we developed a multiplex PCR for detection of the four alleles and applied it directly on clinical specimens. Methods: Subgingival fluid was collected on paper points from three deep pockets in 107 patients with generalized aggressive periodontitis attending the Dental Hospital at The Royal London. Extracted DNA was amplified with primers specific for P. gingivalis 16S rDNA, and with a multiplex of primers developed to differentiate the four variant rag alleles. This work was supported by grant G9900398 from the Medical Research Council (UK). Results: P. gingivalis was detected by 16S rDNA PCR in 72% of patients. rag alleles 1 (W83 type), 2, 3, and 4 (ATCC 33277 type) were detected in 9, 38, 40, and 8% of P. gingivalis-positive patients respectively. Only one patient had more than one allele detected (1 and 3), and in four patients none of the known rag alleles was detected. Conclusion: Multiplex PCR allowed detection of variant rag alleles in clinical specimens without a requirement for microbial culture. The rag alleles of P. gingivalis detected among patients in London with generalized aggressive periodontitis were predominantly rag-2 and rag-3, which differ from rag in the well-characterised laboratory strains W50/W83 and 381/ATCC 33277.