Archives of Microbiology

, Volume 195, Issue 7, pp 473–482 | Cite as

Development of quantitative real-time PCR primers for detecting 42 oral bacterial species

  • Soon-Nang Park
  • Yun Kyong Lim
  • Joong-Ki KookEmail author
Original Paper


In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene (rpoB) for detecting 42 oral bacterial species. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 73–79 strains regarding 73–75 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of 42 bacterial species-specific qPCR primers ranged from 4 to 40 fg below a cycle threshold (C T) value of 35, except Atopobium rimae, Fusobacterium nucleatum, Neisseria meningitidis, and Porphyromonas asaccharolytica which were 400 fg. These results suggest that 42 bacterial species-specific qPCR primers are suitable for applications in epidemiological studies related to oral infectious diseases such as periodontal diseases, endodontic infection, and dental caries.


Quantitative real-time PCR primers  Oral bacteria rpoB 



This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (Grant Number 2009-0076542).

Supplementary material

203_2013_896_MOESM1_ESM.pdf (62 kb)
Supplementary material 1 (PDF 61 kb)
203_2013_896_MOESM2_ESM.pdf (1.8 mb)
Supplementary material 2 (PDF 1830 kb)
203_2013_896_MOESM3_ESM.pdf (1.5 mb)
Supplementary material 3 (PDF 1564 kb)
203_2013_896_MOESM4_ESM.pdf (68 kb)
Supplementary material 4 (PDF 67 kb)


  1. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43:5721–5732PubMedCrossRefGoogle Scholar
  2. Abdeldaim GM, Strålin K, Korsgaard J, Blomberg J, Welinder-Olsson C, Herrmann B (2010) Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. BMC Microbiol 10:310. doi: 10.1186/1471-2180-10-310 PubMedCrossRefGoogle Scholar
  3. Aliyu SH, Marriott RK, Curran MD, Parmar S, Bentley N, Brown NM, Brazier JS, Ludlam H (2004) Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice. J Med Microbiol 53:1029–1035PubMedCrossRefGoogle Scholar
  4. Ashimoto A, Chen C, Bakker I, Slots J (1996) Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 11:266–273PubMedCrossRefGoogle Scholar
  5. Bizhang M, Ellerbrock B, Preza D, Raab W, Singh P, Beikler T, Henrich B, Zimmer S (2011) Detection of nine microorganisms from the initial carious root lesions using a TaqMan-based real-time PCR. Oral Dis 17:642–652. doi: 10.1111/j.1601-0825.2011.01815.x PubMedCrossRefGoogle Scholar
  6. Brito LC, Teles FR, Teles RP, França EC, Ribeiro-Sobrinho AP, Haffajee AD, Socransky SS (2007) Use of multiple-displacement amplification and checkerboard DNA–DNA hybridization to examine the microbiota of endodontic infections. J Clin Microbiol 45:3039–3049PubMedCrossRefGoogle Scholar
  7. Conrads G, Flemmig TF, Seyfarth I, Lampert F, Lutticken R (1999) Simultaneous detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA gene-directed multiplex PCR. J Clin Microbiol 37:1621–1624PubMedGoogle Scholar
  8. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB (2001) Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 39:1553–1558PubMedCrossRefGoogle Scholar
  9. Dolan Thomas J, Hatcher CP, Satterfield DA, Theodore MJ, Bach MC, Linscott KB, Zhao X, Wang X, Mair R, Schmink S, Arnold KE, Stephens DS, Harrison LH, Hollick RA, Andrade AL, Lamaro-Cardoso J, de Lemos AP, Gritzfeld J, Gordon S, Soysal A, Bakir M, Sharma D, Jain S, Satola SW, Messonnier NE, Mayer LW (2011) sodC-based real-time PCR for detection of Neisseria meningitidis. PLoS ONE 6:e19361. doi: 10.1371/journal.pone.0019361 PubMedCrossRefGoogle Scholar
  10. Drancourt M, Raoult D (2002) rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol 40:1333–1338PubMedCrossRefGoogle Scholar
  11. Haffajee AD, Bogren A, Hasturk H, Feres M, Lopez NJ, Socransky SS (2004) Subgingival microbiota of chronic periodontitis subjects from different geographic locations. J Clin Periodontol 31:996–1002PubMedCrossRefGoogle Scholar
  12. Hyvärinen K, Laitinen S, Paju S, Hakala A, Suominen-Taipale L, Skurnik M, Könönen E, Pussinen PJ (2009) Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR. Innate Immun 15:195–204. doi: 10.1177/1753425908101920 PubMedCrossRefGoogle Scholar
  13. Jensen A, Hagelskjaer Kristensen L, Prag J (2007) Detection of Fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time PCR. Clin Microbiol Infect 13:695–701PubMedCrossRefGoogle Scholar
  14. Kato H, Yoshida A, Awano S, Ansai T, Takehara T (2005) Quantitative detection of volatile sulfur compound- producing microorganisms in oral specimens using real-time PCR. Oral Dis 11:67–71 Erratum in: Oral Dis 2005;15:120PubMedCrossRefGoogle Scholar
  15. Khamis A, Raoult D, La Scola B (2004) rpoB gene sequencing for identification of Corynebacterium species. J Clin Microbiol 42:3925–3931PubMedCrossRefGoogle Scholar
  16. Kim HS, Lee DS, Chang YH, Kim MJ, Koh S, Kim J, Seong JH, Song SK, Shin HS, Son JB, Jung MY, Park SN, Yoo SY, Cho KW, Kim DK, Moon S, Kim D, Choi Y, Kim BO, Jang HS, Kim CS, Kim C, Choe SJ, Kook JK (2010) Application of rpoB and zinc protease gene for use in molecular discrimination of Fusobacterium nucleatum subspecies. J Clin Microbiol 48:545–553. doi: 10.1128/JCM.01631-09 PubMedCrossRefGoogle Scholar
  17. Kozarov E, Sweier D, Shelburne C, Progulske-Fox A, Lopatin D (2006) Detection of bacterial DNA in atheromatous plaques by quantitative PCR. Microbes Infect 8:687–693PubMedCrossRefGoogle Scholar
  18. Krieg NR (2001) Identification of prokaryotes. In: Boone DR, Castenholz RW, Garrity GM (eds) Bergey’s manual of systematic bacteriology, vol 1, 2nd edn. Springer-Verlag, New York, pp 33–38CrossRefGoogle Scholar
  19. Kuboniwa M, Amano A, Kimura KR, Sekine S, Kato S, Yamamoto Y, Okahashi N, Iida T, Shizukuishi S (2004) Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol Immunol 19:168–176PubMedCrossRefGoogle Scholar
  20. Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ (2003) New bacterial species associated with chronic periodontitis. J Dent Res 82:338–344PubMedCrossRefGoogle Scholar
  21. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S (2003) 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 39:81–86PubMedCrossRefGoogle Scholar
  22. Nonnenmacher C, Dalpke A, Mutters R, Heeg K (2004) Quantitative detection of periodontopathogens by real-time PCR. J Microbiol Methods 59:117–125PubMedCrossRefGoogle Scholar
  23. Nonnenmacher C, Dalpke A, Rochon J, Flores-de-Jacoby L, Mutters R, Heeg K (2005) Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients. J Periodontol 76:1542–1549PubMedCrossRefGoogle Scholar
  24. Park SN, Park JY, Kook JK (2011) Development of Porphyromonas gingivalis-specific quantitative real-time PCR primers based on the nucleotide sequence of rpoB. J Microbiol 49:315–319. doi: 10.1007/s12275-011-1028-y PubMedCrossRefGoogle Scholar
  25. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE (2001) Bacterial diversity in human subgingival plaque. J Bacteriol 183:3770–3783PubMedCrossRefGoogle Scholar
  26. Price RR, Viscount HB, Stanley MC, Leung KP (2007) Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR. Biofouling 23:203–213PubMedCrossRefGoogle Scholar
  27. Saito D, Coutinho LL, Borges Saito CP, Tsai SM, Höfling JF, Gonçalves RB (2009) Real-time polymerase chain reaction quantification of Porphyromonas gingivalis and Tannerella forsythia in primary endodontic infections. J Endod 35:1518–1524. doi: 10.1016/j.joen.2009.08.005 PubMedCrossRefGoogle Scholar
  28. Saygun I, Kubar A, Sahin S, Sener K, Slots J (2008) Quantitative analysis of association between herpesviruses and bacterial pathogens in periodontitis. J Periodontal Res 43:352–359PubMedCrossRefGoogle Scholar
  29. Severinov K, Mustaev A, Kukarin A, Muzzin O, Bass I, Darst SA, Goldfarb A (1996) Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the beta and beta’ subunits of Escherichia coli RNA polymerase. J Biol Chem 271:27969–27974PubMedCrossRefGoogle Scholar
  30. Yoo SY, Kim KJ, Lim SH, Kim KW, Hwang HK, Min BM, Choe SJ, Kook JK (2005) First isolation of Streptococcus downei from human dental plaques. FEMS Microbiol Lett 249:323–326PubMedCrossRefGoogle Scholar
  31. Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T (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 41:4438–4441PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of DentistryChosun UniversityGwangjuRepublic of Korea

Personalised recommendations