Objective: The purpose of this investigation was to validate a real-time quantitative polymerase chain reaction (PCR) assay in identifying and quantifying Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis from subgingival plaque samples taken from subjects with different periodontal conditions, when compared with conventional cultural procedures.
Patients and methods: Ninety-two adult subjects participated in this study, 32 with periodontitis, 30 with gingivitis and 30 healthy. A pooled subgingival sample was obtained from every patient. Culturing procedures were carried out using standard techniques. For real-time PCR analysis, primers were selected from sequences of the LktC (A. actinomycetemcomitans), Arg-gingipain (P. gingivalis) and BspA antigen (T. forsythensis) genes. Contingency tables were constructed to compare the qualitative results, while quantitative data were evaluated by paired t-test.
Results: A. actinomycetemcomitans was the least frequently recovered species with both techniques. Prevalence of P. gingivalis was low in healthy patients, increased in gingivitis and peaked in periodontitis patients. The frequency of detection of T. forsythensis showed marked differences between culture and PCR, although the same tendency of an increase in prevalence from health to gingivitis and to periodontitis was observed with both methods. Contingency tables demonstrated a good level of agreement between PCR and culture procedures for A. actinomycetemcomitans and P. gingivalis, especially in periodontitis patients. P. gingivalis culture counts were significantly higher than those obtained by PCR. The opposite was true for T. forsythensis, and statistically significant higher counts were obtained by PCR for gingivitis and periodontitis patients.
Conclusion: This study demonstrated a good agreement between the quantitative PCR technology and the culture procedure. The high sensitivity and specificity of the quantitative PCR technology justify its use in epidemiological studies and as an adjunct in clinical diagnosis of periodontal patients.