Detection And Quantification Of Tannerella Forsythia In Subgingival Plaque Samples

Tannerella forsythia was strongly implicated as the major pathogens in chronic periodontitis, but little information has been obtained for the distribution of this bacterium in patients of chronic periodontitis and healthy subjects in China. The quantification of these bacteria in the diseased part of the patient with periodontitis is rarely known. The purpose of this study was to investigate the distribution of Tannerella forsythia in chronic periodontitis patients and healthy subjects, and the relationship between the prevalence of Tannerella forsythia and the clinical parameters. The methods of TaqMan real-time quantitative PCR in detection of Tannerella forsythia and total bacteria in subgingival plaque samples were established and to clarify the relationship between the bacterial load and the periodontal status. The results are as follows:(1) 327 subgingival plaque samples were collected from 108 chronic periodontitis patients and 111 healthy subjects. Tannerella forsythia was detected by 16S rRNA polymerase chain reaction. The prevalence of Tannerella forsythia was higher in diseased sites than in healthy subjects, the prevalence was 56.5% and 10.8% respectively (P<0.001). The distribution of this bacterium in the relative healthy sites was 3.7% in patients, the prevalence of Tannerella forsythia in diseased sites was significant higher than relative healthy sites in patients (P<0.001). The prevalence of Tannerella forsythia in healthy subjects was higher than relative healthy sites in periodontal patients (P<0.05). The results indicate a possible association between periodontal disease and the presence of Tannerella forsythia.(2) Subgingival plaque samples of chronic periodontitis patients were detected. The prevalence of Tannerella forsythia was significantly higher in deeper pockets and more serious clinical attachment loss, spearman rank correlation coefficient was 0.46 and 0.52 respectively (P< 0.001 and P<0.05). The prevalence of Tannerella forsythia in BOP(+) sites was 60.2%,and in BOP(-) sites was 7.3% (P<0.001). The prevalence of Tannerella forsythia was significantly correlated with clinical parameters, suggesting that Tannerella forsythia is closely related to chronic periodontitis in the Chinese population.(3) In order to establish a quantitative assay, cloned plasmids containing the target region of Tannerella forsythia and eubacteria were cloned by using pMD18-T vector and T-A cloning procedures. The recombinant clones were screened by white/blue selection. Insertion was confirmed by agarose gel and Sequence analysis. The results show that this methods of recombinant clones was effective, simplify and rapid.(4) TaqMan real-time polymerase chain reaction assay for Tannerella forsythia and total bacteria. Quantification was performed with species-specific primer/probe, universal primer/probe and serial dilution of plasmid standards. Results showed that the numbers of Tannerella forsythia and total bacteria (P<0.001), the proportion of Tannerella forsythia in subgingival specimens (P<0.05) were significant higher in diseased sites of chronic periodontitis patients than in healthy sites of healthy subjects. In addition, a significant correlation was found between the number of bacteria and various probing depth (P<0.001). Furthermore, a positive correlation was observed between colonization at high levels by Tannerella forsythia and total bacteria and the deeper probing depth. There was no significantly difference between the proportion of Tannerella forsythia in subgingival plaque and probing depth. These results suggest that the numbers of Tannerella forsythia are closely associated with periodontal status.Based on all of the above these findings, we concluded that Tannerella forsythia are closely related to chronic periodontitis in the Chinese population. The numbers of Tannerella forsythia in subgingival plaque samples are closely associated with the occurrence and progression of periodontitis, and demonstrate the broad potential of real-time polymerase chain reaction application on periodontology.