Article | . 2017 Vol. 17, Issue. 1
Analysis of total oral microorganisms in saliva using real-time PCR and colony forming unit

Departments of Dental Hygiene, Kyungdong University1
Departments of Preventive Dentistry, School of Dentistry, Chosun University2
Departments of Preventive Dentistry, School of Dentistry, Dankuk University3
Departments of Dental Hygiene, Hanseo University4

2017.. 13:25


Objectives: The purpose of this study was to compare colony forming unit (CFU) method and multiplex real-time polymerase chain reaction (MRT-PCR) method for accurate quantitative analysis of bacteria. Methods: We compared the CFU method and the MRT-PCR method, which are still used in Korea, for Prevotella intermedius (P. intermedius), a periodontal disease pathogen selected by MRT-PCR, and Streptococcus mutans (S. mutans), a dental caries causative organism. The subjects of this study were 30 patients who visited the C dental hospital. Results: Total microorganisms in MRT-PCR method were significantly higher in both types of bacteria (p<0.05), since DNA of dead bacteria was also analyzed. This was because the periodontal dise(-) anaerobes, and even dead bacteria contain large amounts of toxic substances called LPS in the extracellular membrane, and fimbriae and pili, which are motility structures, still remain as a strong toxic substance in periodontal tissue. Conclusions: Therefore, in terms of the total amount of bacteria found, the MRT-PCR method will be a useful technique for searching all the bacteria in the oral cavity including live bacteria, as well as sterilization.

1. Sastri A. Plaque microorganism and periodontal disease. J Dent Assoc Thai 1977;27(3):88-92.  

2. Yun JH, Park JE, Kim DI, Lee SI, Choi SH, Cho KS, et al. Identification of putative perio-dontal pathogens in Korean chronic periodontitis patients. J Korean Acad Periodontol 2008; 38(2):143-52.  

3. 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(11):4950-4.  

4. Lovegrove JM. Dental plaque revisited: bacteria associated with periodontal disease. J N Z Soc Periodontol 2004;87:7-21.  

5. Jang HS, Kim JY, Kook JK, You SY, Kim HS, Kim SG, et al. Comparison of the prevalence of 4 periodontopathogens in supra-and subgingival plaque of young adults without periodontitis. J Korean Acad Periodontol 2003;33(2):159-66.   

6. Kook JK, Lim SS, You SY, Hwang HK. Antibiotic susceptibility in mutans streptococci and Stretococcus anginosus isolated from dental plaque. J Korean Acad Operative Dent 2004; 29(5):462-9.  

7. Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002;148(Pt 1): 257-66.  

8. Kim JH, You SY, lim SA, Kook JK, Lim SS, Park SH, et al. Identification of putative pathogens in acute endodontic infections by PCR based on 16s rDNA. J Korean Acad Operative Dent 2003;28(2):178-83.  

9. Amicosante M, Richeldi L, Trenti G, Paone G, Campa M, Bietti A, et al. Inactivation of polymerase inhibitors for mycobacterium tuberculosis DNA amplification in sputum by using capture resin. J Clin Microbiol 1995;33(3):629-30.  

10. Johnson SR, Martin DH, Cammarata C, Morse SA. Alterations in sample preparation increase sensitivity of PCR assay for diagnosis of chancroid. J Clin Microbiol 1995;33(4):1036-8.  

11. Mättö J, et al. Detection of Porphyromonas gingivalisfrom saliva by PCR by using a simple sample-processing method. J Clin Microbiol 1998;36(1):157-60.  

12. Kim MJ, et al. Strain-specific PCR primers for the detection of Prevotella intermedia ATCC 49046. Int J Oral Biol 2011;36(2):79-82.  

13. Cho HB. Multiplex real time PCR for simultaneous detection of 6 periodontopathic bacteria. Korean J Microbiol 2013;9:292-6.  

14. Horz HP, Vianna ME, Gomes BP, Conrads G. Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples: general implications and practical use in endodontic antimicrobial therapy. J Clin Microbiol 2005;43(10):5332-7. JCM.43.10.5332-5337.2005  

15. Gouet P, Courcelle E, Stuart DI, Métoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 1999;15(4):305-8.  

16. Löe H, Silness J. Periodontal disease in pregnancy I. Prevalence and severity. Acta Odontol Scand 1963;21:533-51.  

17. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci USA 1996;96(25):14547-52.  

18. Loomer PM. Microbiological diagnostic testing in the treatment of periodontal diseases. Perio-dontol 2000 2004;34:49-56.  

19. Lyons SR, Griffen AL, Leys EJ. Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria. J Clin Microbiol 2000;38(6):2362-5.  

20. Eun AJ, Seoh M, Wong S. Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR. J Virol Methods 2000;87(1-2):151-60.  

21. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M. et al. 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(1):81-6.  

22. Park SN, Park JY, Kook JK. Development of quantitative real-time PCR primers for the detection of Aggregatibacter actinomycetemcomitans. Int J Oral Biol 2011;36(1):1-6.  

23. Dymock D, Weightman AJ, Scully C, Wade WG. Molecular analysis of microflora associated with dentoalveolar abscesses. J Clin Microbiol 1996;34:537-42.   

24. Park SN, Lim YK, Kook JK. Development of quantitative real-time PCR primers for detecting 42 oral bact erial species. Arch Microbiol 2013;195(7):473-82. 013-0896-4  

25. Reardon-Robinson ME, Wu C, Mishra A, Chang C, Bier N, Das A, et al. Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development. Proc Natl Acad Sci USA 2014;111(10):3835-40.  

26. Martin FE, Nadkarni MA, 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(5):1698-704.  

27. Ali RW, Bakken V, Nilsen R, Skaug N. Comparative detection frequency of 6 putative perio-dontal pathogens in Sudanese and Norwegian adult periodontitis patients. J Periodontol 1994; 65(11):1046-52.  

28. Mättö J, Saarela M, Alaliisua S, Oja V, Jousimies-Somer H, Asikainen S. Detection of Porphyromonas gingivalis from Saliva by PCR by using a simple sample-processing method. J Clin Microbiol 1998;36(1):157-60.  

29. Moncla BJ, Braham PH, Persson GR, Page GC, Weinberg A. Direct detection of Porphyromonas gingivalis in Macaca fascicularis dental plaque samples using an oligonucleotide probe. J Periodontol 1994;65(5):398-403.  

30. Rolph HJ, Lennon A, Riggio MP, Saunders WP, MacKenzie D, Coldero L, et al. Molecular identification of microorganisms from endodontic infections. J Clin Microbiol 2001;39(9):3282-9.  

31. Ammann TW, Bostanci N, Belibasekis GN, Thurnheer T. Validation of a quantitative real‐time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species‐specific quantification of an in vitro subgingival biofilm model. J Periodontal Res 2013;48(4):517-26.  

32. Verner C, Lemaitre P, Daniel A, Giumelli B, Lakhssassi N, Sixou M. Carpegen real‐time polymerase chain reaction vs. anaerobic culture for periodontal pathogen identification. Oral Microbiol Immunol 2006;21(6): 341-6.  

33. 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(2):191-9.  

34. Yano A, Kanako N, Ida H, Yamaguchi T, Hanada N. Real-time PCR for quantification of Streptococcus mutans. FEMS microbiol lett 2002;217(1):23-30.  

35. Jervøe-Storm PM, Koltzscher M, Falk W, Dörfler A, Jepsen S. Comparison of culture and real‐time PCR for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples. J Clin Periodontol 2005;32(7):778-3. 051X.2005.00740.x  

36. Moon KH, Kwon H. The influence on the recognition for periodontal care to oral micro-organism changes in dental implant patients. Int J Clin Prev Dent 2016;12(4):221-8.  

37. Kim JB, Paik DI, Moon HS, Choi YJ, Sin SC, Kwon HK, et al. Preventive Dentistry 4ed. Seoul: Komoonsa; 2006: 85-6.  

38. Haffajee AD, Teles RP, Socransky SS. Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions. Oral Microbiol Immunol 2006;21(5):269-82. https://  

online submission
online submission
online submission
online submission
online submission
Sub Menu
Sub Banner
Sub Menu
Sub Banner
Sub Banner
Sub Banner
Sub Banner
Sub Menu
Sub Menu