POLYMERASE CHAIN-REACTION DETECTION OF 8 PUTATIVE PERIODONTAL PATHOGENS IN SUBGINGIVAL PLAQUE OF GINGIVITIS AND ADVANCED PERIODONTITIS LESIONS

A 16S rRNA-based polymerase chain reaction (PCR) detection method was used to determine the prevalence of Actinobacillus actinomycetemcomita ns, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola in subgingival specimens of 50 advanced perio dontitis, 50 adult gingivitis and 50 pediatric gingivitis subjects. Th e optimal PCR conditions were determined for each study species. Agaro se gel electrophoresis of PCR products from each study species reveale d a single band of the predicted size. Restriction enzyme digestion of amplicons confirmed the specificity of the amplification. PCR detecti on limits were in the range of 25-100 cells. No cross-reactivity with other oral microorganisms or nonspecific amplification was observed. T he prevalence by PCR in advanced periodontitis, adult gingivitis and p ediatric gingivitis subjects was 30%, 14% and 14% for A. actinomycetem comitans, 86%, 18% and 8% for B. forsythus, 74%, 52% and 78% for C. re ctus, 80%, 70% and 66% for E. corrodens, 70%, 10% and 14% for P. gingi valis, 58%, 12% and 18% for P, intermedia, 52%, 20% and 22% for P. nig rescens, and 54%, 16% and 16% for T. denticola, respectively. The prev alence was higher in the advanced periodontitis group than in both adu lt gingivitis and pediatric gingivitis for A, actinomycetemcomitans, B . forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. dentic ola at P<0.01, and for E. corrodens at P<0.05. The prevalence of C. re ctus was significantly higher in the advanced periodontitis group than in the adult gingivitis group at P<0.01. Matching results between PCR and culture occurred in 28% (B. forsythus) to 71% (A. actinomycetemco mitans) of the samples; the major discrepancy occurred in the PCR-posi tive/culture-negative category. Matching results between PCR and DNA p robe methods were found in 84% of the subjects (B. forsythus) and 70% (P. gingivalis). Odds ratio analysis revealed statistically significan t positive associations between 17 of the 28 possible combinations (P< 0.01). This study demonstrated the utility of a 16S rRNA-based PCR det ection method for identifying important subgingival microorganisms. Th e results indicated a strong association between the study species and periodontitis. Several previously unreported symbiotic relationships were found between the 8 species tested.