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Detection of Treponema Socranskii Associated with Human Periodontitis by PCR

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Detection of Treponema Socranskii Associated with Human Periodontitis by PCR Microbiol. Immunol., 43(5), 485-490, 1999 Detection of Treponema socranskii Associated with Human Periodontitis by PCR Mitsuo Sakamoto*,', Yasuo Takeuchi2, Makoto Umeda2, Isao Ishikawa2, Yoshimi Benno', and Takashi Nakase' 'Japan Collection of Microorg anisms, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351- 0198, Japan, and "Department of Periodontology, Faculty of'Dentistry, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan Received December 18, 1998; in revised form, February 17, 1999. Accepted February 19, 1999 Abstract: A PCR technique was used to detect and identify Treponema socranskii associated with peri- odontitis. A species-specific forward primer was designed for a variable region within its 16S rRNA gene and was used in conjunction with a conserved reverse primer. This primer pair was tested for specificity against 44 oral bacterial strains. Sensitivity was determined using a serial dilution of T socranskii cells. Amplification products were obtained from all TTsocranskii strains tested, but not from other oral bacte- ria associated with periodontal disease. The detection limit of PCR was 5 T. socranskii cells per PCR. TT socranskii was detected by PCR in subgingival plaque and saliva samples from patients with periodontitis. Key words: Treponema socranskii, Periodontitis, PCR Periodontitis is considered to be a polymicrobial important in epidemiological studies, and in the diag- mixed infection (12). Of the microbial flora proliferating nosis, treatment planning, and monitoring of periodontal in periodontal disease sites, spirochetes are the predom- pathogens. inant bacteria. They can be observed by dark-field or Randomly cloned chromosomal DNA fragments have phase-contrast microscopy in subgingival plaques of been used for the detection and identification of oral patients with periodontitis. However, these bacteria are spirochetes (5). This technique, however, is relatively rarely isolated using traditional culture methods. insensitive. Recently, Moter et al have developed a dot Cultures of oral treponemes are limited because of the blot or in situ hybridization technique which detects unique and complex nutrition and strict anaerobic con- cultivable or so far uncultivable treponemes, including T ditions needed for their growth. The cultivable oral trep- socranskii (13). This method although sensitive, is com- onemes recognized to date include 7 species: Trepone- plicated. PCR-based methods have been used for the ma amylovorum, T denticola, T maltophilum, T medi- detection and identification of periodontal bacteria, such um, T pectinovorum, T socranskii, and T vincentii (2, as Actinobacillus actinomycetemcomitans and Porphy- 18, 19, 22, 24, 25). Among these species, T socranskii is romonas gingivalis (1, 4, 6, 9, 10). In the present study, the most frequently isolated treponeme (7, 11, 13, 16, we describe a PCR-based approach for the detection 19). Its frequency correlated with increasing gingival and identification of T socranskii associated with peri- index scores in both children and adults (11). T socran- odontal disease. skii has also been detected in dental plaque samples The strains used in this study are listed in Table 1. from dogs (17). The prevalence of T socranskii was They include 3 subspecies of T socranskii and other significantly greater in dogs with one or more probing oral bacteria associated with periodontal disease. The T sites >5 mm, than was found in dogs with only healthy periodontal tissues (17). The rapid detection and identi- Abbreviations: CFU, colony-forming unit; DSM, Deutsche fication of T socranskii in subgingival plaque is therefore Sammlung von Mikroorganismen and Zellkulturen, Braun- schweig, Germany; FDC, Forsyth Dental Center, Massachusetts, U.S.A.; JCM, Japan Collection of Microorganisms, RIKEN, *Address correspondence to Dr . Mitsuo Sakamoto, Japan Col- Saitama, Japan; NCTC, National Collection of Type Cultures, lection of Microorganisms, The Institute of Physical and Chem- London, England; PBS, phosphate-buffered saline; PCR, poly- ical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351- merase chain reaction; RFLP, restriction fragment length poly- 0198, Japan. E-mail: [email protected] morphism; RPP, rapidly progressive periodontitis. 485 486 M. SAKAMOTO ET AL Table 1. Bacterial strains used and specificity of Treponema socranskii primer tested °'+ , positive reaction; -, negative reaction. socranskii strains were grown as described previously Kyoto, Japan), 10 l.tl of IO X Ex Taq buffer, 8 µl of (7). All other bacteria were grown under appropriate dNTP mixture (2.5 mm each), and 10 pmol of each culturing conditions. primer. The reaction mixtures were amplified in a A primer for the specific amplification of T socranskii TaKaRa PCR Thermal Cycler MP with the following was selected by aligning the 16S rRNA gene sequences program: 95 C for 3 min, followed by 36 cycles con- (obtained from the DDBJ, EMBL, and GenBank data- sisting of 95 C for 30 sec, 67 C for 30 sec, and 72 C for 1 bases) of the oral bacteria associated with periodontal dis- min, with a final extension period at 72 C for 2 min. ease (Fig. 1) using the CLUSTAL W program (20). The After thermal cycling, 10 tl of the amplified product was sequence of species-specific forward primer TRS207F run on a 1.2% agarose gel, stained with ethidium bro- was 5'-AGG TAG ACA GCG GGA AAG GA-3'(T mide, and visualized using a UV transilluminator. socranskii positions 207 to 226). The sequence of con- To determine the detection limit, a suspension of T served reverse primer 1089R was 5'-TAA CCC AAC socranskii cells was serially diluted and subjected to ACC TCA CGG CA-3'; this sequence covered the con- PCR as described above. served region of the 16S rRNA gene at positions 1108 to To divide T socranskii into 3 subspecies groups and to 1089 (T socranskii numbering). The specificity of confirm the specificity of the TRS207F and 1089R TRS207F primer was checked by the CHECK PROBE primers, PCR products generated with primers were program of the Ribosomal Database Project (8). digested with restriction endonuclease, Ncil or ScrFl Bacterial DNA was prepared according to the fol- (Nippon Gene) according to the manufacturer's instruc- lowing procedure. A 1.5 ml pure culture of the strain was tions. The digested solutions were analyzed by 2% centrifuged. The resulting bacterial pellets were washed agarose gel (MetaPhor Agarose; FMC Bioproducts, twice with phosphate-buffered saline (PBS) and then Rockland, Me., U.S.A.), stained with ethidium bromide, resuspended in 100 µl of PBS buffer. The suspension was and visualized with a UV transilluminator. diluted with 100 gl of 10% Triton X-100, and heated at Supragingival plaque samples were removed with 95 C for 5 min. No further purification of nucleic acids sterile cotton rolls, and subgingival plaque samples were was performed. Five microliters of bulk DNA was sub- taken from four sites in 16 patients (average age: 35.5 jected to PCR in a 100 tl (total volume) reaction mixture years) with rapidly progressive periodontitis (RPP), 10 containing 2.5 U of TaKaRa Ex Taq (Takara Shuzo Co., patients (average age: 53.6 years) with adult periodontitis, NOTES 487 Fig. 1. Sequence of the detection primer for members of the Treponema socranskii group and alignment of the 16S rRNA gene sequences of other Treponema species and oral bacteria associated with periodontal disease. Asterisks indicate nucleotides identical to nucleotides of TTsocranskii. Boldface letters indicate nucleotides identical to the nucleotides of primer TRS207F and the primer regions of other Trep- onema species and oral bacteria associated with periodontal disease. Abbreviations: T socranskii, Treponema socranskii subsp. socranskii; T buccale, Treponema socranskii subsp. buccale; T paredis, Treponema socranskii subsp. paredis. and 6 subjects (average age: 26.6 years) with healthy because the G+C contents of the primer regions of periodontal tissues by inserting sterile paper points into these species are lower than that of T socranskii (e.g., T periodontal pockets (average probing depth: 6.22 mm, socranskii, 55%; T. maltophilum, 45%; B. forsythus, 6.21 mm and 1.53 mm, respectively). After 20 sec, the 35%) (Fig. 1). The sensitivity of the TRS207F primer in paper points were removed and immersed in 1 ml of ster- PCR was determined by using known numbers of T ile distilled water. They were vigorously mixed for 1 min socranskii cells. The detection limit of PCR for T by a vortex, paper points removed, and then centrifuged socranskii was 2 X 103CFU/ml of suspensionin pure culture at 10,000X g for 5 min. The pellets were resuspended in (i.e., approximately 5 bacteria cells in the assay) (Fig. 3). 200 Vl of sterile distilled water, and heated at 100 C for 10 To divide T socranskii into 3 subspecies groups, we min. Five microliters of the supernatant was used as a digested the PCR products with Ncil or ScrFl. The Ncil template for PCR. The whole saliva sample from each digest of the amplicon from T socranskii subsp. socran- individual was collected. The 0.5 ml aliquot of saliva skii gave three visible bands of 141, 284, and 477 bp; sample was diluted 1:2 vol/vol in distilled water and those from T socranskii subsp. buccale gave two visible washed 4 times with distilled water. The washed bacte- bands of 389 and 477 by (the band of 36 by was not visu- rial cell pellet was reconstituted with 0.5 ml of distilled alized); and those from T socranskii subsp. paredis water, and heated at 100 C for 10 min. gave two visible bands of 425 and 477 by (Fig. 4). The results of 16S rRNA gene amplification are These results were expected from the nucleotide shown in Fig. 2. The use of primers TRS207F and sequences of the PCR products. In addition, the ScrFI 1089R resulted in a 902 by PCR fragment from the 3 digest of the amplicon from T socranskii subsp.
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