Optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens

BMC Research Notes

Pieter Deschaght³  Mario Vaneechoutte³  Stefan Vermeulen¹  Els Van Mechelen¹  Bart Saerens³  Reza Miremadi²  Hugo De Bruyn²  Jan Cosyn²  Ellen Decat³ 

[1] Biomedical and Exact Sciences, Faculty of Education, Health&Social Work, University College Ghent, Keramiekstraat 80, Ghent, Belgium;Department of Periodontology and Oral Implantology, Dental School, Faculty of Medicine and Health Sciences, University of Ghent, De Pintelaan 185, Ghent, B-9000, Belgium;Laboratory Bacteriology Research, Department Clinical Chemistry, Microbiology&Immunology, Faculty of Medicine and Health Sciences, University of Ghent, De Pintelaan 185, Ghent, B-9000, Belgium

关键词: Intercycler portability;    Quantification limit;    Specificity;    Periodontal pathogens;    QPCR;   

Others  :  1165085
DOI  :  10.1186/1756-0500-5-664

 received in 2012-07-03, accepted in 2012-11-22,  发布年份 2012

【 摘 要 】

Background

The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans.

Results

Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r² ≥ 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 μl sample in a final volume of 10 μl on the Light Cycler 480.

Conclusions

In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment.

【 授权许可】

   
2012 Decat et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Loe H, Anerud A, Boysen H, Morrison E: Natural history of periodontal disease in man. Rapid, moderate and no loss of attachment in Sri Lankan laborers 14 to 46 years of age. J Clin Periodontol 1986, 13:431-445.
• [2]Slots J, Rams TE: Microbiology of periodontal disease. In Contemporary oral microbiology and immunology. St. Louis: Mosby Year Book; 1992:429-431.
• [3]Collins LM, Dawes C: The surface area of the adult human mouth and thickness of the salivary film covering the teeth and oral mucosa. J Dent Res 1987, 66:1300-1302.
• [4]Paster BJ, Olsen I, Aas JA, Dewhirst FE: The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006, 42:80-87.
• [5]Hommez GM, Verhelst R, Claeys G, Vaneechoutte M, De Moor RJ: Investigation of the effect of the coronal restoration quality on the composition of the root canal microflora in teeth with apical periodontitis by means of T-RFLP analysis. Int Endodont J 2004, 37:819-827.
• [6]Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ: New bacterial species associated with chronic periodontitis. J Dent Res 2003, 82:338-344.
• [7]Moore WE, Moore LV: The bacteria of periodontal diseases. Periodontol 2000 1994, 5:66-77.
• [8]Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE: Bacterial diversity in human subgingival plaque. J Bacteriol 2001, 183:3770-3783.
• [9]Socransky SS, Haffajee AD: Periodontal microbial ecology. Periodontol 2000 2005, 38:135-187.
• [10]Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL: Microbial complexes in subgingival plaque. J Clin Periodontol 1998, 25:134-144.
• [11]Sharawy AM, Sabharwal K, Socransky SS, Lobene RR: A quantitative study of plaque and calculus formation in normal and periodontally involved mouths. J Periodontol 1966, 37:495-501.
• [12]Gendron R, Grenier D, Maheu-Robert L: The oral cavity as a reservoir of bacterial pathogens for focal infections. Microbes Infect 2000, 2:897-906.
• [13]Kolenbrander PE, Palmer RJ Jr, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI: Bacterial interactions and successions during plaque development. Periodontol 2000 2006, 42:47-79.
• [14]Haffajee AD, Socransky SS, Ebersole JL, Smith DJ: Clinical, microbiological and immunological features associated with the treatment of active periodontosis lesions. J Clin Periodontol 1984, 11:600-618.
• [15]Haffajee AD, Socransky SS: Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 1994, 5:78-111.
• [16]Listgarten MA, Lai CH, Evian CI: Comparative antibody titers to Actinobacillus actinomycetemcomitans in juvenile periodontitis, chronic periodontitis and periodontally healthy subjects. J Clin Periodontol 1981, 8:155-164.
• [17]Slots J: Reynolds HS. Genco RJ: Actinobacillus actinomycetemcomitans in human periodontal disease: a cross-sectional microbiological investigation. Infect Immun 1980, 29:1013-1020.
• [18]Tanner A, Maiden MF, Macuch PJ, Murray LL, Kent RL: Microbiota of health, gingivitis, and initial periodontitis. J Clin Periodontol 1998, 25:85-98.
• [19]Van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U: Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002, 29:1023-1028.
• [20]Socransky SS, Haffajee AD: The bacterial etiology of destructive periodontal disease: current concepts. J Periodontol 1992, 63:322-331.
• [21]Booth V, Downes J, Van den Berg J, Wade WG: Gram-positive anaerobic bacilli in human periodontal disease. J Periodontal Res 2004, 39:213-220.
• [22]Choi BK, Park SH, Yoo YJ, Choi SH, Chai JK, Cho KS, Kim CK: Detection of major putative periodontopathogens in Korean advanced adult periodontitis patients using a nucleic acid-based approach. J Periodontol 2000, 71:1387-1394.
• [23]Colombo AP, Teles RP, Torres MC, Souto R, Rosalem WJ, Mendes MC, Uzeda M: Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis. J Periodontol 2002, 73:360-369.
• [24]Herrera D, Roldan S, Gonzalez I, Sanz M: The periodontal abscess (I). Clinical and microbiological findings. J Clin Periodontol 2000, 27:387-394.
• [25]Moore WE, Holdeman LV, Cato EP, Smibert RM, Burmeister JA, Palcanis KG, Ranney RR: Comparative bacteriology of juvenile periodontitis. Infect Immun 1985, 48:507-519.
• [26]Papapanou PN, Baelum V, Luan WM, Madianos PN, Chen X, Fejerskov O, Dahlen G: Subgingival microbiota in adult Chinese: prevalence and relation to periodontal disease progression. J Periodontol 1997, 68:651-666.
• [27]Papapanou PN, Neiderud AM, Papadimitriou A, Sandros J, Dahlen G: “Checkerboard" assessments of periodontal microbiota and serum antibody responses: a case–control study. J Periodontol 2000, 71:885-897.
• [28]Rams TE, Feik D, Slots J: Campylobacter rectus in human periodontitis. Oral Microbiol Immunol 1993, 8:230-235.
• [29]Simonson LG, Robinson PJ, Pranger RJ, Cohen ME, Morton HE: Treponema denticola and Porphyromonas gingivalis as prognostic markers following periodontal treatment. J Periodontol 1992, 63:270-273.
• [30]Okada M, Kawamura M, Oda Y, Yasuda R, Kojima T, Kurihara H: Caries prevalence associated with Streptococcus mutans and Streptococcus sobrinus in Japanese schoolchildren. Int J Paediatr Dent 2012, 22:342-348. Jan 8 [Epub ahead of print]
• [31]Richards W, Amee J, Coll AM, Higgs G: Reasons for tooth extraction in four general dental practices in South Wales. Br Dent J 2005, 198:275-278.
• [32]Cosyn J, Princen K, Miremadi R, Decat E, Vaneechoutte M, De Bruyn H: A double-blind randomized placebo-controlled study on the clinical and microbial effects of an essential oil mouth rinse used by patients in supportive periodontal care. Int J Dent Hyg 2012, 10.1111/idh.12000:10.1111/idh.12000.
• [33]Hyvarinen K, Laitinen S, Paju S, Hakala A, Suominen-Taipale L, Skurnik M, Kononen E, Pussinen PJ: Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR. Innate Immun 2009, 15:195-204.
• [34]Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: Minimum Information for publication of Quantitative real-time PCR Experiments. Clin Chem 2009, 55:611-622.
• [35]Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S: Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol 2003, 39:81-86.
• [36]Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T: 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 2003, 41:4438-4441.
• [37]Martin FE, Nadkarni MA, Jacques NA, Hunter N: Quantitative microbiological study of human carious dentine by culture and real-time PCR: Association of anaerobes with histopathological changes in chronic pulpitis. J Clin Microbiol 2002, 40:1698-1704.
• [38]Suzuki N, Yoshida A, Nakano Y: Quantitative analysis of multi-species oral biofilms by taqman real-time PCR. Clin Med Res 2005, 3:176-185.
• [39]Bartz H, Nonnenmacher C, Bollmann C, Kuhl M, Zimmermann S, Heeg K, Mutters R: Micromonas (Peptostreptococcus) micros: unusual case of prosthetic joint infection associated with dental procedures. Int J Med Microbiol 2005, 294:465-470.
• [40]Shelburne CE, Prabhu A, Gleason RM, Mullally BH, Coulter WA: Quantitation of Bacteroides forsythus in subgingival plaque - Comparison of immunoassay and quantitative polymerase chain reaction. J Microbiol Methods 2000, 39:97-107.
• [41]Morillo JM, Lau L, Sanz M, Herrera D, Martin C, Silva A: Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens. J Clin Periodontol 2004, 31:1054-1060.
• [42]Kuboniwa M, Amono A, Kimura KR, Sekine S, Kato S, Yamamoto Y, Okahashi N, Lida T, Shizukuishi S: Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol Immunol 2004, 19:168-176.
• [43]Yano A, Kaneko N, Ida H, Yamaguchi T, Hanada N: Real-time PCR for quantification of Streptococcus mutans. FEMS Microbiol Lett 2002, 217:23-30.
• [44]Signat B, Roques C, Poulet P, Duffaut D: Role of Fusobacterium nucleatum in periodontal health and disease. Curr Issues Mol Biol 2011, 13:25-35.
• [45]Hoebeeck J, Speleman F, Vandesompele J: Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes. New Jersey: Humana Press; 2007:205-226. [Methods in Molecular Biology. Volume 353]
• [46]Persson RG, Hitti J, Verhelst R, Vaneechoutte M, Persson RE, Hirschi R, Weibel M, Rothen M, Temmerman M, Paul K, Eschenbach D: The vaginal microflora in relation to gingivitis. BMC Infect Dis 2009, 9:6. BioMed Central Full Text
• [47]Haffajee AD, Yaskell T, Torresyap G, Teles R, Socransky SS: Comparison between polymerase chain reaction-based and checkerboard DNA hybridization techniques for microbial assessment of subgingival plaque samples. J Clin Periodontol 2009, 36:642-649.
• [48]Boutaga K, van Winkelhoff AJ, Vandenbroucke-Grauls CM, Savelkoul PH: Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples. J Clin Microbiol 2003, 41:4950-4954.
• [49]Boutaga K, van Winkelhoff AJ, Vandenbroucke-Grauls CM, Savelkoul PH: Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR. FEMS Immunol Med Microbiol 2005, 45:191-199.
• [50]Boutaga K, Savelkoul PH, Winkel EG, Van Winkelhoff AJ: Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction. J Periodontol 2007, 78:79-86.
• [51]Rams TE, Listgarten MA, Slots J: Utility of 5 major putative periodontal pathogens and selected clinical parameters to predict periodontal breakdown in patients on maintenance care. J Clin Periodontol 1996, 23:346-354.