Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43:5721–5732

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 53:1029–1035

    Article  PubMed  CAS  Google Scholar 

  • 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 11:266–273

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 45:3039–3049

    Article  PubMed  CAS  Google Scholar 

  • 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 37:1621–1624

    PubMed  CAS  Google Scholar 

  • 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 39:1553–1558

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Drancourt M, Raoult D (2002) rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol 40:1333–1338

    Article  PubMed  CAS  Google Scholar 

  • 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 31:996–1002

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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 13:695–701

    Article  PubMed  CAS  Google Scholar 

  • 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;15:120

    Article  PubMed  Google Scholar 

  • Khamis A, Raoult D, La Scola B (2004) rpoB gene sequencing for identification of Corynebacterium species. J Clin Microbiol 42:3925–3931

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Kozarov E, Sweier D, Shelburne C, Progulske-Fox A, Lopatin D (2006) Detection of bacterial DNA in atheromatous plaques by quantitative PCR. Microbes Infect 8:687–693

    Article  PubMed  CAS  Google Scholar 

  • 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 33–38

    Chapter  Google Scholar 

  • 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 19:168–176

    Article  PubMed  CAS  Google Scholar 

  • Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ (2003) New bacterial species associated with chronic periodontitis. J Dent Res 82:338–344

    Article  PubMed  CAS  Google Scholar 

  • 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 39:81–86

    Article  PubMed  CAS  Google Scholar 

  • Nonnenmacher C, Dalpke A, Mutters R, Heeg K (2004) Quantitative detection of periodontopathogens by real-time PCR. J Microbiol Methods 59:117–125

    Article  PubMed  CAS  Google Scholar 

  • 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 76:1542–1549

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 183:3770–3783

    Article  PubMed  CAS  Google Scholar 

  • 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 23:203–213

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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 43:352–359

    Article  PubMed  CAS  Google Scholar 

  • 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 271:27969–27974

    Article  PubMed  CAS  Google Scholar 

  • 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 249:323–326

    Article  PubMed  CAS  Google Scholar 

  • 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 41:4438–4441

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

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).

Author information

Authors and Affiliations

  1. Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of Dentistry, Chosun University, 375 Seo-suk Dong, Dong-Gu, Gwangju, 501-759, Republic of Korea

    Soon-Nang Park, Yun Kyong Lim & Joong-Ki Kook

Authors
  1. Soon-Nang Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun Kyong Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joong-Ki Kook
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joong-Ki Kook.

Additional information

Communicated by Erko Stackebrandt.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, SN., Lim, Y.K. & Kook, JK. Development of quantitative real-time PCR primers for detecting 42 oral bacterial species. Arch Microbiol 195, 473–482 (2013). https://doi.org/10.1007/s00203-013-0896-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-013-0896-4

Keywords

Search

Navigation