Detection and quantification of oral treponemes in subgingival plaque by real-time PCR

Abstract

Oral treponemes have been associated with periodontal diseases. We developed a highly sensitive and specific method to detect and quantify cultivable oral treponemes (Treponema denticola, Treponema vincentii, and Treponema medium) in 50 subgingival plaque samples from 13 healthy subjects as well as 37 patients with periodontal diseases using real-time PCR assays with specific primers and a TaqMan probe for each 16S rRNA sequence. The specificity for each assay was examined by using DNA specimens from various treponemal and other bacterial species. The TaqMan real-time PCR was able to detect from 10(3) to 10(8) cells of the oral treponemes, with correlation coefficients as follows: T. denticola, 0.984; T. vincentii, 0.991; and T. medium, 0.984. The frequencies of occurrence of these three oral treponemes in subgingival plaque samples were as follows: T. denticola, 68.0%; T. vincentii, 36.0%; and T. medium, 48.0%. In addition, the number of T. denticola, T. vincentii, and T. medium cells in plaque samples detected by real-time PCR ranged from 3 to 15,184, 1 to 447, and 1 to 7,301 cells/pg of plaque DNA, respectively. Increased numbers of T. denticola cells were detected in plaque samples from deep periodontal pockets, and T. medium was also detected in deep pockets. On the other hand, T. vincentii was mainly found in shallow pockets. These results suggest that various oral treponemes are associated with the formation of each stage of periodontal disease.

FIG. 1.

Electrophoresis evaluation of PCR products amplified with primers for T. denticola (A), T. vincentii (B), T. medium (C), and total treponemes (D) and with a ubiquitous primer (E). Lanes: M, molecular size marker (a 100-bp DNA ladder); 1, T. denticola; 2, T. vincentii; 3, T. medium; 4, T. socranskii; 5, T. phagedenis; 6, T. pectinovorum; 7, P. gingivalis; 8, P. nigrescens; 9, A. actinomycetemcomitans; 10, E. coli; 11, F. nucleatum; 12, S. mutans; 13, S. oralis; and 14, S. salivarius. The expected sizes are noted by arrows.

FIG. 2.

Standard curves generated by analysis of a known number of T. denticola (A), T. vincentii (B), and T. medium (C) cells by real-time PCR. Linearity is observed from 10³ to 10⁸ cells of these treponemes (correlation coefficients, 0.984 for T. denticola, 0.991 for T. vincentii, and 0.984 for T. medium).