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Quantitative real‐time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model
Author(s) -
Sánchez M. C.,
Marín M. J.,
Figuero E.,
LlamaPalacios A.,
León R.,
Blanc V.,
Herrera D.,
Sanz M.
Publication year - 2014
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/jre.12073
Subject(s) - propidium monoazide , biofilm , microbiology and biotechnology , fusobacterium nucleatum , antimicrobial , isopropyl alcohol , porphyromonas gingivalis , streptococcus mutans , chemistry , bacteria , in vitro , real time polymerase chain reaction , periodontal pathogen , isopropyl , biology , biochemistry , genetics , organic chemistry , gene
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. actinomycetemcomitans , 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. Biofilms were disrupted and PMA added (final concentration of 100 μ m ). 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 antimicrobial agent. Hence, this PMA ‐ qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.