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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\u2010time polymerase chain reaction (<jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content>) combined with propidium monoazide (<jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content>), developed for the selective quantification of viable <jats:italic>P.\u00a0gingivalis<\/jats:italic>,<jats:italic> A.\u00a0actinomycetemcomitans<\/jats:italic>,<jats:italic> F.\u00a0nucleatum<\/jats:italic> and total bacteria in an <jats:italic>in vitro<\/jats:italic> biofilm model after antimicrobial treatment.<\/jats:p><\/jats:sec><jats:sec><jats:title>Material and Methods<\/jats:title><jats:p><jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content>\u2010<jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content> method was tested in an <jats:italic>in vitro<\/jats:italic> biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30\u00a0min to isopropyl alcohol by immersion. Biofilms were disrupted and <jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content> added (final concentration of 100\u00a0\u03bc<jats:sc>m<\/jats:sc>). After <jats:styled-content style=\"fixed-case\">DNA<\/jats:styled-content> isolation, <jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content> 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>When <jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content> was used in the presence of viable target bacterial cells, no statistically significant inhibition of <jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content> amplification was detected (<jats:italic>p<\/jats:italic>\u00a0&gt;\u00a00.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, <jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content> resulted in a significant total reduction of <jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content> amplification of about 4 log<jats:sub>10<\/jats:sub>. <jats:italic>P.\u00a0gingivalis<\/jats:italic> showed a vitality reduction in the biofilm of 3 log<jats:sub>10<\/jats:sub>, while <jats:italic>A.\u00a0actinomycetemcomitans<\/jats:italic> and <jats:italic>F.\u00a0nucleatum<\/jats:italic> showed a 2 log<jats:sub>10<\/jats:sub> reduction.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>These results demonstrate the efficiency of <jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content> for differentiating viable and dead <jats:italic>P.\u00a0gingivalis<\/jats:italic>,<jats:italic> A.\u00a0actinomycetemcomitans<\/jats:italic> and <jats:italic>F.\u00a0nucleatum<\/jats:italic> cells<jats:italic>,<\/jats:italic> as well as total bacteria, in an <jats:italic>in vitro<\/jats:italic> biofilm model, after being exposed to an antimicrobial agent. Hence, this <jats:styled-content style=\"fixed-case\">PMA<\/jats:styled-content>\u2010<jats:styled-content style=\"fixed-case\">qPCR<\/jats:styled-content> method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.<\/jats:p><\/jats:sec>","DOI":"10.1111\/jre.12073","type":"journal-article","created":{"date-parts":[[2013,4,15]],"date-time":"2013-04-15T04:50:54Z","timestamp":1366001454000},"page":"20-28","source":"Crossref","is-referenced-by-count":47,"title":["Quantitative real\u2010time <scp>PCR<\/scp> combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an <i>in vitro<\/i> subgingival biofilm model"],"prefix":"10.1111","volume":"49","author":[{"given":"M. C.","family":"S\u00e1nchez","sequence":"first","affiliation":[{"name":"Research Laboratory Faculty of Dentistry, Complutense University of Madrid Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. 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