Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples

Abstract

Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of P. gingivalis in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the P. gingivalis 16S rRNA gene. By the real-time PCR assay, as few as 1 CFU of P. gingivalis could be detected. Subgingival plaque samples from 259 adult patients with severe periodontitis were analyzed. P. gingivalis was detected in 111 (43%) of the 259 subgingival plaque samples by culture and in 138 (53%) samples by PCR. The sensitivity, specificity, and positive and negative predictive values of the real-time PCR were 100, 94, 94, and 100%, respectively. We conclude that real-time PCR confirms the results of quantitative culture of P. gingivalis and offers significant advantages with respect to the rapidity and sensitivity of detection of P. gingivalis in subgingival plaque samples.

FIG. 1.

Quantification of P. gingivalis amplification. Serial 10-fold dilutions (a to g, with 650,000 to 0.65 CFU/reaction mixture) of P. gingivalis DNA were amplified with primers P.g.F and P.g.R and detected with TaqMan probe P.g.P. ΔRn, change in fluorescence intensity. The correlation coefficient (R) for the C_t values was 0.999.

FIG. 2.

Scatter plot showing the differences and correlations between the real-time PCR and the anaerobic culture method. Data for P. gingivalis-positive versus P. gingivalis-negative samples by both methods (n = 11 and n = 121, respectively) fall close to the line of equivalence (R² = 0.977). Samples that were PCR positive and culture negative fall near the x axis (n = 27). Samples which were negative by both methods are shown with an arrow (n = 121). A second linearity coefficient was calculated only for the quantitative results for the 111 samples positive by culture and PCR (R² = 0.366).