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dc.contributor.authorSoto Muñoz, Lourdes
dc.contributor.authorTeixidó i Espasa, Neus
dc.contributor.authorUsall i Rodié, Josep
dc.contributor.authorViñas Almenar, Inmaculada
dc.contributor.authorCrespo Sempere, Ana
dc.contributor.authorTorres Sanchis, Rosario
dc.date.accessioned2017-05-26T13:00:09Z
dc.date.issued2014
dc.identifier.issn0168-1605
dc.identifier.urihttp://hdl.handle.net/10459.1/59708
dc.description.abstractDilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface.
dc.description.sponsorshipThis research was supported by the national project RTA2009-00053-00-00 (Plan Nacional de I+D Ministerio de Ciencia e Innovación, Spanish Government) and the National Council of Science and Technology of México (CONACyT) for scholarship 198363 (Soto-Muñoz L.). We are also grateful to Cristina Solsona for her excellent technical assistance.
dc.format.mimetypeapplication/pdf
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relationMICINN/PN2008-2011/RTA2009-00053-00-00
dc.relation.isformatofReproducció del document publicat a: https://doi.org/10.1016/j.ijfoodmicro.2014.04.011
dc.relation.ispartofInternational Journal of Food Microbiology, 2014, vol. 180, p. 49-55
dc.rights(c) Elsevier, 2013
dc.subjectBiocontrol
dc.subjectFruit
dc.subjectQuantitative method
dc.subjectViable cells
dc.titleDevelopment of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges
dc.typeinfo:eu-repo/semantics/article
dc.date.updated2017-05-26T13:00:10Z
dc.identifier.idgrec021078
dc.type.versioninfo:eu-repo/semantics/publishedVersion
dc.rights.accessRightsinfo:eu-repo/semantics/restrictedAccess
dc.identifier.doihttps://doi.org/10.1016/j.ijfoodmicro.2014.04.011
dc.date.embargoEndDate10000-01-01


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