Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model S anchez MC, Mar  ın MJ, Figuero E, Llama-Palacios A, Le  on R, Blanc V, Herrera D, Sanz M. Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model. 2013; doi: 10.1111/jre.12073. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objectives: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. ac- tinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. Material and Methods: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Bio- films were disrupted and PMA added (final concentration of 100 lM). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. Results: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log 10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log 10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log 10 reduction. Conclusion: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an M. C. S  anchez 1 , M. J. Mar  ın 1 , E. Figuero 1 , A. Llama-Palacios 1 , R. Le on 2 , V. Blanc 2 , D. Herrera 3 , M. Sanz 3 1 Research Laboratory, Faculty of Dentistry, Complutense University of Madrid, Spain, 2 Dentaid SA, Barcelona, Spain and 3 Etiology and Therapy of Periodontal Disease Research Group, Faculty of Dentistry, Complutense University of Madrid, Spain Dr Mariano Sanz, Section of Periodontology, Faculty of Odontology, Plaza Ram on y Cajal s/n, Ciudad Universitaria, Madrid 28040, Spain Tel: +(34) 913941901 Fax: +(34) 913941910 e-mail: marianosanz@odon.ucm.es Key words: Aggregatibacter actinomycetemcomitans; antimicrobial agents; Fusobacterium nucleatum; oral biofilm; Porphyromonas gingivalis; propidium monoazide; quantitative real-time PCR Accepted for publication February 02, 2013 J Periodont Res 2013 All rights reserved © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12073