Multiplex Real-Time PCR for Simultaneous Detection of 6 Periodontopathic Bacteria

Multiplex Real-Time PCR을 이용하여 6종의 주요 잇몸질환 유발 미생물을 동시에 검출하는 기법

  • Received : 2013.09.09
  • Accepted : 2013.09.26
  • Published : 2013.09.30


This study utilized an analysis method for detecting six microorganisms, such as Actinobacillus actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Tannerella forsythus, Treponema denticola, and Prevotella intermedia, triggering periodontal disease, using multiplex real-time polymerase chain reaction (PCR). The analysis including internal control was made by dividing the six species into two groups using four fluorescence dyes, and it was verified that there was no interference or cross-reaction between the target species and different kinds of oral microbial species. Qualitative and quantitative analyses were conducted on each microorganism in various samples, such as saliva and the plaque, using the multiplex real-time PCR and comparative analysis between periodontitis patients and healthy people, revealing obvious differences between them.


multiplex real-time PCR;periodontopathic bacteria;plaque;saliva


  1. Asai, Y., Jinno, T., Igarashi, H., Ohyama, Y., and Ogawa, T. 2002. Detection and quantification of oral treponemes in subgingival plaque by real-time PCR. J. Clin. Microbiol. 40, 3334-3340.
  2. Chamberlain, J.S. and Chamberlain, J.R. 1994. Optimization of multiplex PCRs, pp. 38-46. In Mullis, K.B., Ferre, F., and Gibbs, R.A. (eds.), The polymerase chain reaction. Birkhauser, Boston, Mass., USA.
  3. Flemmig, T.F., Rudiger, S., Hofmann, U., Schmidt, H., Plaschke, B., Stratz, A., Klaiber, B., and Karch, A. 1995. Identification of Actinobacillus actinomycetemcomitansin subgingival plaque by PCR. J. Clin. Microbiol. 33, 3102-3105.
  4. Garcia, L., Tercero, J.C., Legido, B., Ramos, J.A., Alemany, J., and Sanz, M. 1998. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Porphyromona gingivalis by multiplex PCR. J. Periodont. Res. 33, 59-64.
  5. Haffajee, A.D. and Socransky, S.S. 1994. Microbial etiological agents of destructive periodontal diseases. Periodontology 5, 78–111.
  6. Han, X.Y., Tarrand, J.J., and Rice, D.C. 2005. Oral Campylobacter species involved in extraoral abscess: a report of three cases. J. Clin. Microbiol. 43, 2513-2515.
  7. Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H., and Vogt, P.H. 1997. Multiplex PCR: critical parameters and step-by-step protocol. BioTechniques 23, 504–511.
  8. Simon D.T. and Rudney, J.D. 1999. Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis. J. Clin. Microbiol. 37, 3504-3508.
  9. Simonson, L.G., McMahon, K.T., Childers, D.W., and Morton, H.E. 1992. Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population. Oral. Microbiol. Immunol. 7, 111-112.
  10. Takamatsu, N., Yano, K., He, T., Umeda, M., and Ishikawa, I. 1999. Effect of initial periodontal therapy on the frequency of detecting Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. J. Periodontol. 70, 574-580.
  11. Tran, S.D. and Rudney, J.D. 1996. Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. J. Clin. Microbiol. 34, 2674-2678.
  12. Wahlfors, J., Meurman, J.H., Vaisanen, P., Alakuijala, P., Korhonen, A., Torkko, H., and Janne, J. 1995. Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method. J. Dent. Res. 74, 1796-1801.
  13. Wolff, L.F., Aeppli, D.M., Pihlstrom, B.L., Anderson, L., Stoltenberg, J.L., Osborn, J.B., Hardie, N.A., Shelburne, C.E., and Fisher, G.E. 1993. Natural distribution of 5 bacteria associated with periodontal disease. J. Clin. Periodontol. 20, 699-706.
  14. Zambon, J.J. 1996. Periodontal diseases: microbial factors. Ann. Periodontol. 1, 879-925.

Cited by

  1. Comparison of quantitative detection of periodontal pathogens before and after scaling by real-time polymerase chain reaction vol.15, pp.6, 2015,