TODENTJ-25-03-2019_TODENTJ-2-49 1553504988 Bentham Open doi@benthamopen.net Bentham Open The Open Dentistry Journal TODENTJ 1874-2106 3 11 2008 2 1 S.S. Kirakodu M. Govindaswami M.J. Novak J.L. Ebersole K.F. Novak Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples. 3 11 2008 49 55 TODENTJ-2-49 1 10.2174/crossmark_policy opendentistryjournal.com benthamopen.com true Peer Reviewed Single blind Checked with iThenticate 2007-11-29 2008-2-4 2008-3-11 http://creativecommons.org/licenses/by-nc/3.0/ 10.2174/1874210600802010049 1553504988 https://opendentistryjournal.com/VOLUME/2/PAGE/49/ https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.pdf https://opendentistryjournal.com/contents/volumes/V2/TODENTJ-2-49/TODENTJ-2-49.xml Novak MJ. Clinical Periodontology. 9. Philadelphia PA: Saunders WB 2002; pp. 64-73. Haake SK, Newman MG, Nisengard RJ, Sanz M. Clinical Periodontology. 9. Philadelphia PA: Saunders WB 2002; pp. 96-112. J Med Microbiol Doungudomdacha S 49 861 2000 Doungudomdacha S, Rawlinson A, Douglas CW. Enumeration ofPorphyromonas gingivalis Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method J Med Microbiol 2000; 49 : 861-74. J Clin Microbiol Griffen AL 36 3239 1998 Griffen AL, Becker MR, Lyons SR. Prevalence ofPorphyromonas gingivalis and periodontal health status J Clin Microbiol 1998; 36 : 3239-42. Periodontol 2000 Loomer PM 34 49 2004 Loomer PM. Microbiological diagnostic testing in the treatment of periodontal diseases Periodontol 2000 2004; 34 : 49-56. J Microbiol Methods Nonnenmacher C 59 117 2004 Nonnenmacher C, Dalpke A, Mutters R, Heeg K. Quantitative detection of periodontopathogens by real-time PCR J Microbiol Methods 2004; 59 : 117-25. J Med Microbiol Sakamoto M 53 563 2004 Sakamoto M, Huang Y, Ohnishi M, Umeda M, Ishikawa I, Benno Y. Changes in oral microbial profiles after periodontal treatment as determined by molecular analysis of 16S rRNA genes J Med Microbiol 2004; 53 : 563-71. Microbiol Immunol Sakamoto M 45 39 2001 Sakamoto M, Takeuchi Y, Umeda M, Ishikawa I, Benno Y. Rapid detection and quantification of five periodontopathic bacteria by real-time PCR Microbiol Immunol 2001; 45 (1) : 39-44. Periodontol Slots J 20 82 2000 Slots J, Ting M. Actinobacillus actinomycetemcomitansand Porphyromonas gingivalis in human periodontal disease: occurrence and treatment Periodontol 2000; 20 : 82-121. J Clin Periodontol Dzink JL 15 316 1998 Dzink JL, Socransky SS, Haffajee AD. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases J Clin Periodontol 1998; 15 : 316-23. J Periodontol Conrads G 67 994 1996 Conrads G, Mutters R, Fischer J, Brauner A, Lutticken R, Lampert F. PCR reaction and dot-blot hybridization to monitor the distribution of oral pathogens within plaque samples of periodontally healthy individuals J Periodontol 1996; 67 : 994-1003. Biotechniques Socransky SS 17 788 1994 Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Lewin AE. “Checkerboard” DNA-DNA hybridizations Biotechniques 1994; 17 : 788-92. Oral Microbiol Immunol Ashimoto A 11 266 1996 Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions Oral Microbiol Immunol 1996; 11 : 266-73. J Dent Res Meurman JH 76 1376 1997 Meurman JH, Wahlfors J, Korhonen A, et al. Identification of Bacteriodes forsythus in subgingival dental plaque with the aid of a rapid PCR method J Dent Res 1997; 76 : 1376-80. Clin Infect Dis Slots J 20 S304 1995 Slots J, Ashimoto A, Flynn MJ, Li G, Chen C. Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplification with the polymerase chain reaction Clin Infect Dis 1995; 20 (suppl 2) : S304-7. J Clin Microbiol Asai Y 40 3334 2002 Asai Y, Gino T, Igarashi H, Ohyama Y, Ogawa T. Detection and quantification of oral treponemes in subgingival plaque by real-time PCR J Clin Microbiol 2002; 40 (9) : 3334-40. J Clin Microbiol Lyons SR 38 2362 2000 Lyons SR, Griffen AL, Leys EJ. Quantitative real-time PCR forPorphyromonas gingivalis and total bacteria J Clin Microbiol 2000; 38 : 2362-5. J Clin Periodontol Morillo JM 31 1054 2004 Morillo JM, Lau L, Sanz M, Herrera D, Martin CM, Silva A. Quantitative real-time polumerase chain reaction based on single copy gene sequence for detection of periodontal pathogens J Clin Periodontol 2004; 31 : 1054-60. Microbiology Nadkarni MA 148 257 2002 Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primer set Microbiology 2002; 148 : 257-2566. J Periodontol Nonnenmacher C 76 1542 2005 Nonnenmacher C, Dalpke A, Rochon J, Flores-de-Jacoby L, Mutters R, Heeg K. Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients J Periodontol 2005; 76 : 1542-9. J Clin Periodontol Socransky SS 29 260 2002 Socransky SS, Smith C, Haffajee AD. Subgingival microbial profiles in refractory periodontal disease J Clin Periodontol 2002; 29 : 260-8. FEMS Microbiol Lett Yano A 217 23 2002 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. J Clin Microbiol Yoshida A 41 863 2003 Yoshida A, Suzuki N, Nakano Y, Oho T, Kawada M, Koga T. Development of a 5' fluorogenic nuclease-based real-time PCR assay for quantitative detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis J Clin Microbiol 2003; 41 : 863-6. Oral Microbiol Immunol Dibart S 113 30 1998 Dibart S, Skobe Z, Snapp KR, Socransky SS, Smith C, Kent R. Identification of bacterial species on or in crevicular epithelial cells from healthy and periodontally diseases patients using DNA-DNA hybridization Oral Microbiol Immunol 1998; 113 : 30-5. J Clin Microbiol Sanz M 31 1034 2004 Sanz M, Lau L, Herrera D, Morillo JM, Silva A. Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques a review J Clin Microbiol 2004; 31 : 1034-47. J Clin Microbiol Martin FE 40 1698 2002 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 : 1698-704. J Bacteriol Blakemore RP 128 616 1976 Blakemore RP, Canale-Parola E. Arginine catabolism by Treponema denticola J Bacteriol 1976; 128 (2) : 616-22. J Virol Methods Broberg EK 112 53 2003 Broberg EK, Nygardas M, Salmi AA, Hukkanen V. Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by lightCycler quantitative real-time PCR J Virol Methods 2003; 112 : 53-65. Appl Environ Microbiol Fey A 70 3618 2004 Fey A, Eichler S, Flavier S, Christen R, Hofle M, Guzman A. Establishment of real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism Appl Environ Microbiol 2004; 70 (6) : 3618-23. J Endod Baumgartner JC 25 413 1999 Baumgartner JC, Watkins BJ, Bae KS, Xia T. Association of black-pigmented bacteria with endodontic infections J Endod 1999; 25 : 413-5. J Endod Conrads G 23 433 1997 Conrads G, Gharbia SE, Gulabivala K, Lampert F, Shah HN. The use of 16S rDNA directed PCR for the detection of endodontopathogenic bacteria J Endod 1997; 23 : 433-8. Oral Microbiol Immunol Goncharoff P 8 105 1993 Goncharoff P, Figurski DH, Stevens RH, Fine DH. Identification of Actinobacillus actinomycetemcomitans Polymerase chain reaction amplification of LktA-specific sequences Oral Microbiol Immunol 1993; 8 : 105-10. FEMS Immunol Med Microbiol Maeda H 39 81 2003 Maeda H, Fujimoto C, Haruki Y, et al. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia, tetQ gene and total bacteria FEMS Immunol Med Microbiol 2003; 39 : 81-6. Oral Microbiol Immunol Kuboniwa M 19 168 2004 Kuboniwa M, Amano A, Kimura KR, et al. Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes Oral Microbiol Immunol 2004; 19 : 168-76. Genome Res Heid CA 6 986 1996 Heid CA, Stevens J, Livak KJ, Williams M. Real time quantitative PCR Genome Res 1996; 6 : 986-94. Biochemica Betzl G 1 22 2000 Betzl G, Seller M, Eichner C, et al. Reproducibility of PCR on the LightCycler system Biochemica 2000; 1 : 22-6. Periodontol Socransky SS 38 135 2005 Socransky SS, Haffajee AD. Periodontal microbial ecology Periodontol 2005; 38 : 135-87.