Quantitative real‐time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model

• DOI: 10.1111/jre.12073
• Publication Date: 2013-04-15T04:50:54Z
• Authors: M. C. Sánchez; M. J. Marín; E. Figuero; A. Llama‐Palacios; R. León; V. Blanc; D. Herrera; M. Sanz
• Keywords: Propidium monoazide; Fusobacterium nucleatum; Isopropyl alcohol; Periodontal pathogen; Isopropyl

Background and Objectives Differentiation of live dead cells is an important challenge when using molecular diagnosis for microbial identification. This particularly relevant bacteria have been exposed to antimicrobial agents. The objective this study was test a method quantitative real‐time polymerase chain reaction ( qPCR ) combined with propidium monoazide PMA ), developed the selective quantification viable P. gingivalis , A. actinomycetemcomitans F. nucleatum total in vitro biofilm model after treatment. Material Methods ‐ tested model, isopropyl alcohol as agent. Matured biofilms were 1, 5, 10 30 min by immersion. Biofilms disrupted added (final concentration 100 μ m ). After DNA isolation, carried out specific primers probes target bacteria. differentiation analysis variance. Results When used presence bacterial cells, no statistically significant inhibition amplification detected p > 0.05 all cases). Conversely, immersion biofilm, resulted reduction about 4 log . showed vitality 3 while 2 reduction. Conclusion These results demonstrate efficiency differentiating well bacteria, being Hence, may be useful studying effect agents aimed at oral biofilms.