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Oral Biology pp 191-202 | Cite as

Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR)

  • Mª José Marin
  • Elena Figuero
  • David Herrera
  • Mariano SanzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1537)

Abstract

The quantitative polymerase chain reaction (qPCR) is a variant of PCR aimed to detect and quantify a targeted DNA molecule through the addition of probes labeled with fluorescent molecules that emit fluorescence within each amplification cycle, what results in fluorescence values proportional to the amount of accumulated PCR product. This chapter presents the detailed procedures for quantification of different periodontal pathogens (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, and Fusobacterium spp.) using qPCR. It also includes the description of the most frequent problems encountered and how to solve them. In addition, a detailed protocol for multiplex qPCR to detect and quantify P. gingivalis and A. actinomycetemcomitans is included.

Key words

Quantitative PCR (qPCR) TaqMan Primers Probe Multiplex qPCR Aggregatibacter actinomycetemcomitans Porphyromonas gingivalis Tannerella forsythia Campylobacter rectus Fusobacterium spp 

References

  1. 1.
    Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43:5721–5732CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Consensus Report (1996) Periodontal diseases: pathogenesis and microbial factors. Ann Periodontol 1:926–932CrossRefGoogle Scholar
  3. 3.
    Forner L, Larsen T, Kilian M, Holmstrup P (2006) Incidence of bacteremia after chewing, tooth brushing and scaling in individuals with periodontal inflammation. J Clin Periodontol 33:401–407CrossRefPubMedGoogle Scholar
  4. 4.
    Hartzell JD, Torres D, Kim P, Wortmann G (2005) Incidence of bacteremia after routine tooth brushing. Am J Med Sci 329:178–180CrossRefPubMedGoogle Scholar
  5. 5.
    Crasta K, Daly CG, Mitchell D, Curtis B, Stewart D, Heitz-Mayfield LJ (2009) Bacteraemia due to dental flossing. J Clin Periodontol 36:323–332CrossRefPubMedGoogle Scholar
  6. 6.
    Figuero E, Sanchez-Beltran M, Cuesta-Frechoso S, Tejerina JM, del Castro JA, Gutierrez JM, Herrera D, Sanz M (2011) Detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction. J Periodontol 82:1469–1477CrossRefPubMedGoogle Scholar
  7. 7.
    Oteo A, Herrera D, Figuero E, O'Connor A, Gonzalez I, Sanz M (2010) Azithromycin as an adjunct to scaling and root planing in the treatment of Porphyromonas gingivalis-associated periodontitis: a pilot study. J Clin Periodontol 37:1005–1015CrossRefPubMedGoogle Scholar
  8. 8.
    Herrera D, Alonso B, Leon R, Roldan S, Sanz M (2008) Antimicrobial therapy in periodontitis: the use of systemic antimicrobials against the subgingival biofilm. J Clin Periodontol 35(8 Suppl):45–66CrossRefPubMedGoogle Scholar
  9. 9.
    Sanz M, Lau L, Herrera D, Morillo JM, Silva A (2004) 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 Periodontol 31:1034–1047CrossRefPubMedGoogle Scholar
  10. 10.
    Lockhart PB, Brennan MT, Sasser HC, Fox PC, Paster BJ, Bahrani-Mougeot FK (2008) Bacteremia associated with toothbrushing and dental extraction. Circulation 117:3118–3125CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Maestre JR, Mateo M, Sanchez P (2008) Bacteriemia secundaria a procedimientos odontologicos periodontales. [Bacteremia after periodontal procedures]. Rev Esp Quimioter 21:153–156PubMedGoogle Scholar
  12. 12.
    Lafaurie GI, Mayorga-Fayad I, Torres MF, Castillo DM, Aya MR, Baron A, Hurtado PA (2007) Periodontopathic microorganisms in peripheric blood after scaling and root planing. J Clin Periodontol 34:873–879CrossRefPubMedGoogle Scholar
  13. 13.
    Kinane DF, Riggio MP, Walker KF, MacKenzie D, Shearer B (2005) Bacteraemia following periodontal procedures. J Clin Periodontol 32:708–713CrossRefPubMedGoogle Scholar
  14. 14.
    Daly CG, Mitchell DH, Highfield JE, Grossberg DE, Stewart D (2001) Bacteremia due to periodontal probing: a clinical and microbiological investigation. J Periodontol 72:210–214CrossRefPubMedGoogle Scholar
  15. 15.
    Morillo JM, Lau L, Sanz M, Herrera D, Martin C, Silva A (2004) Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens. J Clin Periodontol 31:1054–1060CrossRefPubMedGoogle Scholar
  16. 16.
    Boutaga K, van Winkelhoff AJ, Vandenbroucke-Grauls CMJE, Savelkoul PHM (2003) Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples. J Clin Microbiol 41:4950–4954CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lau L, Sanz M, Herrera D, Morillo JM, Martin C, Silva A (2004) Quantitative real-time polymerase chain reaction versus culture: a comparison between two methods for the detection and quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in subgingival plaque samples. J Clin Periodontol 31:1061–1069CrossRefPubMedGoogle Scholar
  18. 18.
    Doungudomdacha S, Rawlinson A, Walsh TF, Douglas CW (2001) Effect of non-surgical periodontal treatment on clinical parameters and the numbers of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans at adult periodontitis sites. J Clin Periodontol 28:437–445CrossRefPubMedGoogle Scholar
  19. 19.
    Figuero E, Lindahl C, Marin MJ, Renvert S, Herrera D, Ohlsson O, Wetterling T, Sanz M (2014) Quantification of periodontal pathogens in vascular, blood, and subgingival samples from patients with peripheral arterial disease or abdominal aortic aneurysms. J Periodontol 85:1182–1193CrossRefPubMedGoogle Scholar
  20. 20.
    Mackay J (2007) Introduction to kinetic (real-time) PCR. Methods Mol Biol 353:167–176PubMedGoogle Scholar
  21. 21.
    Mackay J, Landt O (2007) Real-time PCR fluorescent chemistries. Methods Mol Biol 353:237–261PubMedGoogle Scholar
  22. 22.
    Bolivar AMR, Agustina; Garcia-Lugo, Pablo (2014) PCR and PCR-multiplex: critical parameters and standardization protocol. Avances en Biomedicina 3:23–25Google Scholar
  23. 23.
    Squeri R, La Fauci V, Cannavo G, Lo Giudice G, Sindoni L (2006) Identification of the microorganisms responsible for periodontopathy by Multiplex RT-PCR. J Prev Med Hyg 47:142–145PubMedGoogle Scholar
  24. 24.
    Morikawa M, Chiba T, Tomii N, Sato S, Takahashi Y, Konishi K, Numabe Y, Iwata K, Imai K (2008) Comparative analysis of putative periodontopathic bacteria by multiplex polymerase chain reaction. J Periodontal Res 43:268–274CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mª José Marin
    • 1
  • Elena Figuero
    • 1
    • 2
    • 3
  • David Herrera
    • 2
    • 3
  • Mariano Sanz
    • 2
    • 3
    Email author
  1. 1.Oral Research Laboratory, Faculty of OdontologyUniversity ComplutenseMadridSpain
  2. 2.Etiology and Therapy of Periodontal Diseases (ETEP) Research GroupUniversity ComplutenseMadridSpain
  3. 3.Department of Periodontology, Faculty of DentistryUniversity Complutense of MadridMadridSpain

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