F. periodonticum is a gram negative anaerobic bacterium that was first isolated from advanced periodontitis lesions (1). F. periodonticum lives as a commensal, preferentially colonizing the gingival sulcus and periodontal pockets but being capable of opportunistic pathogenicity in the mouth and other body sites (2). The relationship between F. periodonticum and periodontal disease is unclear because there is no rapid and sensitive tool for the detection of F. periodonticum.
PCR is the most sensitive and valuable method to use in epidemiological studies for determining the major causative bacteria in periodontal disease (3, 4). Recently, a putative F. nucleatum subsp. nucleatum–specific DNA probe, Fu4, which could potentially identify F. nucleatum subsp. nucleatum at the subspecies level by restriction fragment length polymorphism, was cloned (5). The probe Fu4 (1268 bp, GenBank accession number AY072632) is composed of the 5′-end of the partial 5′dUTPase gene (273 bp out of 441) and the 3′-end of the partial zinc protease gene (878 bp out of 1227 bp). The two genes are separated by three base pair nucleotides. Therefore, F. nucleatum subsp. nucleatum-specific PCR primers were designed based on the nucleotide sequence of Fu4. However, these PCR primers were able to amplify the target genes from all clinical isolates of F. periodonticum and F. nucleatum tested at low annealing temperatures (unpublished data). These reports suggest that 5′dUTPase and zinc protease genes are well conserved in F. periodonticum and F. nucleatum. In this study, F. periodonticum-specific PCR primers were developed based on the nucleotide sequences of zinc protease and 5′dUTPase genes.
The following bacterial strains were used: F. periodonticum ATCC 33693T, F. nucleatum subsp. nucleatum ATCC 25586T, F. nucleatum subsp. nucleatum ATCC 23726, F. nucleatum subsp. fusiforme ATCC 51190T, F. nucleatum subsp. animalis ATCC 50051T, F. nucleatum subsp. polymorphum ATCC 10953T, F. nucleatum subsp. vincentii ATCC 49256T, F. simiae ATCC 16798T, F. canifelinum ATCC 49733T, F. naviforme CCUG 50052T, F. alocis ATCC 35896T, F. sulci ATCC 35585T, F. russii ATCC 25533T, Aggregatibacter actinomycetemcomitans ATCC 33384T, Porphyromonas gingivalis ATCC 33277T, Prevotella intermedia ATCC 25611T, Prevotella nigrescens ATCC 33563T, Tannerella forsythia ATCC 43037T, Streptococcus mutans ATCC 25175T, Streptococcus mitis KCTC 3556T, and Streptococcus anginosus KCTC 33397T. All reference strains were supplied by the ATCC, Manassas, VA, USA, CCUG (Göteborg, Sweden) or KCTC (Daejeon, Korea). All clinical strains of F. periodonticum (KCOM 1259, KCOM 1261, KCOM 1262, KCOM 1263, KCOM 1277, KCOM 1321, KCOM 1282, KCOM 1283, KCOM 2305) and F. nucleatum (KCOM 1231, KCOM 1232, KCOM 1248, KCOM 1250, KCOM 1253, KCOM 1258, KCOM 1272, KCOM 1279, KCOM 1323, KCOM 1325) were identified at the species level using the comparison method of 16S rDNA, RNA polymerase β–subunit gene (rpoB) and zinc protease gene sequences (6).
Fusobacteria strains were grown in Schaedler broth (Difco Laboratories, Detroit, MI, USA). P. intermedia, P. nigrescens, T. forsythia and P. gingivalis were cultured in TSB (Difco Laboratories) supplemented with 0.5% yeast extract, 0.05% cysteine HCl-H2O, 0.5 mg/mL hemin and 2 μg/mL vitamin K1. A. actinomycetemcomitans was grown in a medium composed of TSB (Difco Laboratories) supplemented with 0.6% yeast extract, 5% horse serum, 75 μg/mL bacitracin and 5 μg/mL vancomycin (Sigma, St. Louis, MO, USA). The above species were grown at 37°C in an anaerobic chamber under 10% H2, 5% CO2 and 85% N2. The Streptococci strains were grown in brain heart infusion (Difco Laboratory) at 37°C under aerobic conditions.
The bacterial genomes were prepared using a G-spin Genomic DNA Extraction kit (iNtRON, Seoul, Korea) according to the manufacturer's instructions. DNA concentrations were determined by measuring the OD at 260 and 280 nm using UV-spectrophotometry (Ultrospec 2000, Pharmacia Biotech, Cambridge, UK).
A pair of PCR primers were designed based on the nucleotide sequence of the partial 5′dUTPase and zinc protease genes of F. periodonticum ATCC 33693T (GenBank accession numbers GQ275001 and GQ275033), F. nucleatum ATCC 25586T (GenBank accession numbers GQ275001 and GQ275046), and F. nucleatum ATCC 10953T (GenBank accession number GQ275000 and GQ275045). The oligonucleotide forward and reverse primers, which were specific to F. periodonticum, were designed using the PRIMERSECT program (DNAStar Lasergene 8.0, DNAStar, Madison, WI, USA.). Table 1 lists the nucleotide sequences of the two selected primers.
|Primer names||Oligonucleotide sequences (5′→3′)||Amplicon size (bp)||Annealing temperature (°C)|
|Fp-F3||ACC TTA TCA AGA CTT ATT ATT TC||665||55|
|Fp-R2||TCA AAC TCT ATY TCA GGA ACA A|
|Fp-F1||ACT TGT ATC TTT TTC ATT TAT||785||50|
|Fp-R2||TCA AAC TCT ATY TCA GGA ACA A|
PCR was performed to validate primer specificity and determine their sensitivity. Specificity was evaluated by examining 12 Fusobacterium species or subspecies, 8 representatives of different oral species, and 19 clinical isolates (9 F. periodonticum and 10 F. nucleatum strains). To test the sensitivity of PCR, the detection limit was defined as the smallest amount of bacterial genomic DNA that could be detected by PCR. This was determined by a serial dilution of a genomic DNA mixture from F. periodonticum ATCC 33693T. The sensitivities ranged from 4 ng to 4 fg using 10-fold dilutions.
PCR was performed using an AccuPower PCR PreMix (Bioneer, Daejeon, Korea) containing 5 nmol of each deoxynucleoside triphosphate, 0.8 μmol KCl, 0.2 μmole Tris-HCl (pH 9.0), 0.03 μmol MgCl2, and 1 unit of Taq DNA polymerase. The bacterial genomic DNA and 20 pmols of each primer were added to a PCR PreMix tube. The PCR was carried out in a final volume of 20 μL. The PCR reaction was run for 30 cycles on a Peltier thermal cycler (Model PTC-200 DNA Engine, MJ Research, Waltham, MA, USA) under the following conditions: initial denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 30 s, primer annealing at the appropriate temperature (Table 1) for 45 s, and extension at 72°C for 30 s; followed by a final extension at 72°C for 5 min. A 2 μL aliquot of the reaction mixture was then analyzed by 1.5% agarose gel electrophoresis in a Tris-acetate buffer (0.04 M Tris-acetate, 0.001 M EDTA, [pH 8.0]) at 100 V for 30 min. The amplification products were stained with ethidium bromide and visualized using a UV transilluminator.
The data obtained from the specificity test showed that the two pairs of PCR primers, Fp-F3/Fp-R2 and Fp-F1/Fp-R2, were species-specific for F. periodonticum against the type strains of representative oral bacteria (Fig. 1). The specificity of the PCR primers for F. periodonticum was confirmed using the genomic DNA of nine clinical isolates against 10 clinical isolates of F. nucleatum, which is genetically close to F. periodonticum (Fig. 2). The Fp-F3/Fp-R2 and Fp-F1/Fp-R2 primers sets were able to detect 4 pg or 40 pg of the genomic DNA of F. periodonticum ATCC 33693T, respectively (Fig. 3.). The annealing temperature and sensitivity of the Fp-F3/Fp-R2 primers were higher than Fp-F1/Fp-R2 primer set. Therefore, the Fp-F3/Fp-R2 pair of primers is strongly recommended for detection of F. periodonticum.
In DNA-DNA hybridization, a comparison of the 16S rDNA sequence is one of the gold standard methods for classifying bacteria at the species level (7). However, in molecular epidemiological studies, it is hard to identify all the clinical strains from dental plaque samples at the species level using the 16S rDNA sequence comparison method. Therefore, a PCR method with species-specific PCR primers designed based on the nucleotide sequences of 16S rDNA is generally used for this purpose. The homology of the 16S rDNA sequences between F. nucleatum and F. periodonticum showed 99.4–99.8% similarity, which is too high to generate F. periodonticum-specific PCR primers (6). The RpoB gene was used to classify bacteria at the species level (8–10). Similarly to the 16S rRNA gene (16S rDNA), the nucleotide sequences of the rpoB are well conserved among bacterial species (6). Recently, the rpoB of F. periodonticum was cloned, sequenced and used as a control to discriminate F. nucleatum at the subspecies level (6). According to this data, the homology of rpoB between F. nucleatum and F. periodonticum was relatively lower than that of 16S rDNA. However, F. periodonticum-specific PCR primers based on the nucleotide of rpoB were not available (data not shown).
The OAT of the two sets of PCR primers, as determined by gradient PCR (data not shown), were 55°C and 50°C, respectively (Table 1). OAT is defined as the highest temperature at which the PCR products can be amplified only from the genomic DNA of the target bacterial strain and at which the amount of PCR product is highest (11). The OATs for the Fp-F3/Fp-R2 and Fp-F1/Fp-R2 primers, as suggested by the PrimerSelect program, were 46.2°C and 45.1°C, respectively. In our experience, the OATs for the PCR primers designed by the PrimerSelect program are generally 5°C to 12°C higher than the predicted temperatures (4, 11). Therefore, it is strongly recommended that the OAT of the PCR primers be determined by gradient PCR.
To our knowledge, this is the first report of development of F. periodonticum-specific PCR primers. These results suggest that the two sets of PCR primers, Fp-F3/Fp-R2 and Fp-F1/Fp-R2, are highly sensitive and applicable to the detection of F. periodonticum in molecular epidemiological studies of periodontal diseases.