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

Sánchez Beltrán, María Del Carmen; Marín Cuenda, María José; Figuero Ruiz, Elena; Llama Palacios, María Arantxazu; León, Rubén; Blanc, Vanesa; Herrera González, David; Sanz Alonso, Mariano

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

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Official URL

https://doi.org/10.1111/jre.12073

Publication date

2014

Authors

Sánchez Beltrán, María Del Carmen

Marín Cuenda, María José

Figuero Ruiz, Elena

Llama Palacios, María Arantxazu

León, Rubén

Blanc, Vanesa

Herrera González, David

Sanz Alonso, Mariano

Publisher

Willey

Citations

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URI

https://hdl.handle.net/20.500.14352/94503

Citation

Sánchez MC, Marín MJ, Figuero E, Llama-Palacios A, León 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. J Periodontal Res. 2014 Feb;49(1):20-8.

Abstract

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 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 log10. P. gingivalis showed a vitality reduction in the biofilm of 3 log10, while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 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.