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Division of Community Oral Health Science, Kyushu Dental College, Kitakyushu 803-8580
Department of Oral Bacteriology, Ohu University School of Dentistry, Koriyama 963-8611, Japan
ABSTRACT
Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.
TEXT
A real-time PCR assay has been developed for quantifying DNA. This method allows easy, rapid, quantitative detection of microorganisms in clinical samples (7, 21). Quantitative analysis with the identification of periodontopathic bacteria is important for the diagnosis, therapeutic evaluation, and risk assessment of periodontal disease (8, 14, 17, 18, 20, 22).
Of the periodontopathic bacteria, Prevotella species form a major portion of the microflora found in human gingival crevices in patients with periodontal disease (10). Prevotella intermedia and Prevotella nigrescens were recently distinguished from each other (19). The two previously recognized genotypes of P. intermedia were elevated to species rank as P. intermedia, corresponding to genotype I, and P. nigrescens, corresponding to genotype II. (6). Thus, identification of the species-specific region between genotypically similar species is essential for the detection of closely related strains, especially in oral biofilms. However, these organisms share over 94% identity on the basis of their 16S rRNA sequences (12). Furthermore, whole-genome sequence information for P. nigrescens is not yet available. Therefore, we used the genomic subtractive hybridization technique to identify species-specific regions that distinguish P. intermedia and P. nigrescens. This technique has successfully identified genomic differences between closely related strains (1). This report first describes the identification of a species-specific sequence for P. nigrescens using subtractive hybridization, the design of a P. nigrescens primer-probe set using this technique, and the quantification of four representative oral black-pigmented Prevotella species using a TaqMan real-time PCR assay.
The bacterial strains used in this study are listed in Table 1. The P. intermedia, P. nigrescens, Prevotella melaninogenica, and Prevotella loescheii strains were cultured anaerobically (10% CO2, 10% H2, 80% N2) at 37°C in GAM broth (Nissui Medical Co., Tokyo, Japan) supplemented with hemin (5 μg/ml) and menadione (0.5 μg/ml). The isolation of genomic DNA and treatment of subgingival plaque samples from patients were performed as previously described (16, 21). Briefly, genomic DNA was isolated and purified using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) in accordance with the manufacturer's instructions for gram-negative bacteria. Subgingival plaque samples were obtained, using a sterile endodontic paperpoint, from the periodontal pockets of 10 patients (four females and six males) with a mean age of 54.2 years (range, 32 to 68 years). The sampled pockets had a mean probing depth of 4.3 ± 1.9 mm (range, 2 to 10 mm). The paperpoint was transferred into 200 μl of cell lysis buffer (1.0% Triton X-100, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA) and boiled at 100°C for 5 min, and the supernatant was used as the PCR template (16).
The oligonucleotide primers and probes designed using Primer Express software (Applied Biosystems, Foster City, Calif.) are listed in Table 2. The P. intermedia-, P. melaninogenica-, and P. loescheii-specific primers and probes were designed from the phoC (4, 5), phyA (2), and plaA (9) genes, respectively, which encode acid phosphatase of P. intermedia, hemolysin of P. melaninogenica, and an adhesion precursor of P. loescheii, respectively. The specificities of the primers and probes were initially analyzed using BLAST (3) on the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/). Then, the specificities of the primers were confirmed using conventional PCR with various bacteria (Table 1) under the follow conditions: 94°C for 5 min, followed by 25 cycles of 94°C for 15 s, 50°C (for P. melaninogenica) or 55°C (for P. intermedia and P. loescheii) for 30 s, and 72°C for 1 min. The specificities of the PCR products and probes were confirmed using DNA sequencing and dot blot analysis with digoxigenin-labeled probes, respectively (data not shown).
To design species-specific primers and TaqMan probes for P. nigrescens ATCC 25261, genomic subtractive hybridization was performed as previously described (13, 15). Chromosomal DNA of P. intermedia ATCC 25611 was used as the driver DNA for P. nigrescens ATCC 25261. The oligonucleotide adaptors used in this study are listed in Table 2. The second-round PCR products were digested with Sau3AI, cloned into BamHI-digested pBluescript II SK+ (Stratagene, La Jolla, Calif.), and then used to transform Escherichia coli DH5 (Takara Bio Co., Shiga, Japan). The nucleotide sequences of 15 randomly selected clones from a DNA bank containing approximately 150 colonies were determined using ABI PRISM 3100 (Applied Biosystems). The inserted fragments of genomic subtracted DNA varied from 150 to 1,000 bp and had different sequences. Three clones (Pn21, Pn29, and Pn32) encoded the putative type II restriction enzyme HpaII of Bacteroides spp., and three identical clones (Pn1, Pn4, and Pn15; Pn4 and Pn15 are the same fragment) had high homology with an unknown functional protein of P. intermedia ST17 (GenBank accession number NC 003441). Seven clones (Pn11, Pn20, Pn22, Pn36, Pn38, Pn27, and Pn49; Pn36 and Pn38 are the same fragment) were identical to the 16S rRNA gene of P. nigrescens, and two clones (Pn18 and Pn51) shared no significant homology. Table 3 shows the characteristics of three representative fragments of the 15 clones.
The P. nigrescens-specific primers and probe were designed from the Sau3AI DNA fragments (Pn18) using genomic subtractive hybridization (Table 3), and the specificities of the DNA sequences between the primers were first confirmed using blastn and then confirmed as previously described (Table 1).
The amplification and detection of DNA using real-time PCR were performed with the LightCycler system (Roche Diagnostics, Mannheim, Germany). For each real-time PCR, 20 μl of a mixture containing 5 μl of lysed cells, 10x LightCycler FastStart DNA Master Hybridization Probes (Roche Diagnostics), each sense and antisense primer at a concentration of 500 nM, and 200 nM TaqMan probe was placed in each capillary. The following DNA amplification conditions were used: 1 cycle of 95°C for 10 min, followed by 60 cycles of 95°C for 10 s and 55°C for 30 s.
Using these primers and probes, we developed a TaqMan real-time PCR assay to quantify four oral black-pigmented Prevotella species. A standard curve was plotted for each primer-probe set using the threshold cycle (Ct) values obtained by amplifying successive 10-fold dilutions of a known concentration of DNA, and the correlations between Ct values and CFU counts were obtained (Fig. 1). The assay was capable of detecting bacterial CFU linearly over a range from 1.59 to 1.59 x 106 for P. intermedia, 1.00 to 1.00 x 105 for P. melaninogenica, 1.44 to 1.44 x 106 for P. loescheii, and 1.3 x 10 to 1.3 x 107 for P. nigrescens. The presence of PCR inhibitors in dental plaque was assessed using the fluorescence levels of serial dilutions of four lysed Prevotella species. Lysates spiked with approximately 10 μg (wet weight) of dental plaque negative for the four Prevotella species showed no inhibitors (data not shown).
Using this real-time PCR assay, we determined the numbers of P. intermedia, P. melaninogenica, P. loescheii, and P. nigrescens bacteria in subgingival plaque from 10 individuals (Table 4). The bacterial numbers ranged from 0 to 2.39 x 102 cells for P. intermedia, 0 to 2.71 x 10 cells for P. melaninogenica, 0 to 7.31 x 102 cells for P. loescheii, and 0 to 4.20 x 10 cells for P. nigrescens per mixture. In this study, three P. intermedia, three P. melaninogenica, five P. loescheii, and four P. nigrescens strains were detected in the 10 individuals. The detection rate is consistent with a previous study (11). In this study, however, no significant quantitative or symbiotic relationships between these organisms in relation to pocket depth were observed (data not shown). Further detailed studies to clarify the quantitative and symbiotic relationships between these black-pigmented Prevotella species in the oral microflora are now in progress.
Our results indicate that the combined approach of real-time PCR quantification and genomic subtractive hybridization is useful for constructing species-specific primer-probe sets for closely related strains and for clarifying the quantitative relationships of Prevotella species in oral biofilms. There are many bacterial species in oral biofilms, and this method is very useful for analyzing specific bacteria therein. We are currently focusing on the symbiotic relationships among oral Prevotella species in periodontal sites.
ACKNOWLEDGMENTS
We thank Masaaki Okamoto, Department of Oral Microbiology, Tsurumi University, for useful suggestions regarding this research.
This investigation was supported by grants in aid (B) 14370700 (to T.T.) and (C) 13672164 (to T.A.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by a research grant from the Clinical Research Foundation (to A.Y.); and by research fellowships from the Japan Society for the Promotion of Science for Young Scientists (to S.N.).
REFERENCES
Akopyants, N. S., A. Fradkov, L. Diatchenko, J. E. Hill, P. D. Siebert, S. A. Lukyanov, E. D. Sverdlov, and D. E. Berg. 1998. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95:13108-13113.
Allison, H. E., and J. D. Hillman. 1997. Cloning and characterization of a Prevotella melaninogenica hemolysin. Infect. Immun. 65:2765-2771.
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
Ansai, T., S. Awano, X. Chen, T. Fuchi, T. Arimoto, S. Akifusa, and T. Takehara. 1998. Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia. FEBS Lett. 428:157-160.
Chen, X., T. Ansai, S. Awano, T. Iida, S. Barik, and T. Takehara. 1999. Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia. J. Bacteriol. 181:7107-7114.
Frandsen, E. V., K. Poulsen, and M. Kilian. 1995. Confirmation of the species Prevotella intermedia and Prevotella nigrescens. Int. J. Syst. Bacteriol. 45:429-435.
Heid, C. A., J. Stevens, K. J. Livak, and P. M. Williams. 1996. Real time quantitative PCR. Genome Res. 6:986-994.
Kuboniwa, M., A. Amano, K. R. Kimura, S. Sekine, S. Kato, Y. Yamamoto, N. Okahashi, T. Iida, and S. Shizukuishi. 2004. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol. Immunol. 19:168-176.
Manch-Citron, J. N., J. Allen, M. Moos, Jr., and J. London. 1992. The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift. J. Bacteriol. 174:7328-7336.
Moore, W. E., L. V. Holdeman, E. P. Cato, R. M. Smibert, J. A. Burmeister, K. G. Palcanis, and R. R. Ranney. 1985. Comparative bacteriology of juvenile periodontitis. Infect. Immun. 48:507-519.
Okuda, K., Y. Fukumoto, and I. Takazoe. 1988. Enumeration of cultivable black-pigmented Bacteroides species in human subgingival dental plaque and fecal samples. Oral Microbiol. Immunol. 3:28-31.
Paster, B. J., F. E. Dewhirst, I. Olsen, and G. J. Fraser. 1994. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria. J. Bacteriol. 176:725-732.
Perrin, A., X. Nassif, and C. Tinsley. 1999. Identification of regions of the chromosome of Neisseria meningitidis and Neisseria gonorrhoeae which are specific to the pathogenic Neisseria species. Infect. Immun. 67:6119-6129.
Sakamoto, M., Y. Takeuchi, M. Umeda, I. Ishikawa, and Y. Benno. 2001. Rapid detection and quantification of five periodontopathic bacteria by real-time PCR. Microbiol. Immunol. 45:39-44.
Suzuki, N., Y. Nakano, A. Yoshida, Y. Yamashita, and Y. Kiyoura. 2004. Real-time TaqMan PCR for quantifying oral bacteria during biofilm formation. J. Clin. Microbiol. 42:3827-3830.
Suzuki, N., Y. Nakano, Y. Yoshida, D. Ikeda, and T. Koga. 2001. Identification of Actinobacillus actinomycetemcomitans serotypes by multiplex PCR. J. Clin. Microbiol. 39:2002-2005.
Suzuki, N., A. Yoshida, T. Saito, M. Kawada, and Y. Nakano. 2004. Quantitative microbiological study of subgingival plaque by real-time PCR shows correlation between levels of Tannerella forsythensis and Fusobacterium spp. J. Clin. Microbiol. 42:2255-2257.
Tanaka, M., Y. Yamamoto, M. Kuboniwa, A. Nonaka, N. Nishida, K. Maeda, K. Kataoka, H. Nagata, and S. Shizukuishi. 2004. Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor. Microbes Infect. 6:1078-1083.
van Steenbergen, T. J., C. J. Bosch-Tijhof, M. D. Petit, and U. Van der Velden. 1997. Intra-familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens. J. Periodontal Res. 32:345-350.
Yoshida, A., M. Kawada, N. Suzuki, Y. Nakano, T. Oho, T. Saito, and Y. Yamashita. 2004. TaqMan real-time polymerase chain reaction assay for the correlation of Treponema denticola numbers with the severity of periodontal disease. Oral Microbiol. Immunol. 19:196-200.
Yoshida, A., N. Suzuki, Y. Nakano, M. Kawada, T. Oho, and T. Koga. 2003. Development of a 5' nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus. J. Clin. Microbiol. 41:4438-4441.
Yoshida, A., N. Suzuki, Y. Nakano, T. Oho, M. Kawada, and T. Koga. 2003. Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. J. Clin. Microbiol. 41:863-866.