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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dc.contributor.authorSánchez Beltrán, María Del Carmen
dc.contributor.authorMarín Cuenda, María José
dc.contributor.authorFiguero Ruiz, Elena
dc.contributor.authorLlama Palacios, María Arantxazu
dc.contributor.authorLeón, Rubén
dc.contributor.authorBlanc, Vanesa
dc.contributor.authorHerrera González, David
dc.contributor.authorSanz Alonso, Mariano
dc.date.accessioned2024-01-22T16:58:58Z
dc.date.available2024-01-22T16:58:58Z
dc.date.issued2014
dc.description.abstractBackground 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.
dc.description.departmentDepto. de Especialidades Clínicas Odontológicas
dc.description.facultyFac. de Odontología
dc.description.refereedTRUE
dc.description.statuspub
dc.identifier.citationSánchez 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.
dc.identifier.doi10.1111/jre.12073
dc.identifier.essn1600-0765
dc.identifier.issn0022-3484
dc.identifier.officialurlhttps://doi.org/10.1111/jre.12073
dc.identifier.urihttps://hdl.handle.net/20.500.14352/94503
dc.issue.number1
dc.journal.titleJournal of Periodontal Research
dc.language.isoeng
dc.page.final28
dc.page.initial20
dc.publisherWilley
dc.rights.accessRightsrestricted access
dc.subject.cdu616.31-022
dc.subject.keywordAggregatibacter actinomycetemcomitans
dc.subject.keywordAntimicrobial agents
dc.subject.keywordFusobacterium nucleatum
dc.subject.keywordPorphyromonas gingivalis
dc.subject.keywordPropidium monoazide
dc.subject.keywordQuantitative real-time PCR
dc.subject.keywordOral biofilm
dc.subject.ucmPeriodoncia
dc.subject.ucmMicrobiología médica
dc.subject.unesco3201.03 Microbiología Clínica
dc.titleQuantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model
dc.typejournal article
dc.type.hasVersionVoR
dc.volume.number49
dspace.entity.typePublication
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