RESEARCH ARTICLE


Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm



S.S. Kirakodu*, M. Govindaswami, M.J. Novak, J.L. Ebersole , K.F. Novak
Center for Oral Health Research, College of Dentistry, University of Kentucky College of Dentistry, Lexington, USA


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© Kirakoduet al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License(http://creativecommons.org/licenses/by-nc/3.0/),which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* Address correspondence to this author at the Center for Oral Health Research, 414 Health Sciences Research Building, 1095 VA Drive, University of Kentucky, Lexington, KY 40536-0305, USA; Tel: 859-323-0281; Fax: 859-257-6566; E-mail: sskira2@uky.edu


Abstract

Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.

Key Word: qPCR, oral bacteria, biofilms.