Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production

The microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in gr...

Descripción completa

Detalles Bibliográficos
Autores: Truchado, Pilar, Gil, Maria Isabel, Kostic, Tanja, Allende, Ana
Tipo de recurso: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2016
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/343181
Acceso en línea:http://hdl.handle.net/10261/343181
Access Level:acceso abierto
Palabra clave:Viable cells
Microbial sampling program
Indicator microorganisms
Cultivation-based techniques
Propidium monoazide
id ES_67f56bfcd1e065bc18dbe5fe88bbd221
oai_identifier_str oai:digital.csic.es:10261/343181
network_acronym_str ES
network_name_str España
repository_id_str
spelling Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary productionTruchado, PilarGil, Maria IsabelKostic, TanjaAllende, AnaViable cellsMicrobial sampling programIndicator microorganismsCultivation-based techniquesPropidium monoazideThe microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in groundwater samples. This study focuses on the optimization, validation and application of a qPCR method combined with propidium monoazide (PMA) treatment to exclude DNA from dead cells. A first screening consisting of six primer sets targeting single and multi-copy of E. coli were tested to evaluate the sensitivity of the assay. After that, four primer sets were selected, combined with PMA treatment and their capacity to distinguish viable cells when combined with a background of dead cells was assessed. A primer set targeting the 23S rRNA gene was 10-fold more sensitive than the rest of primers, enabling the detection of low concentrations of viable E. coli cells. This assay also exhibited good repeatability and reproducibility, which indicates the robustness of the method. Optimized and validated PMA-qPCR was used to enumerate E. coli in environmental samples including irrigation water and fresh produce. The results were compared with the levels quantified using qPCR and cultivation-based techniques. Counts of E. coli using plate count assay were significantly lower than the levels obtained by molecular techniques (PMA-qPCR and qPCR) in both irrigation water and fresh produce. E. coli PMA-qPCR enumeration method showed similar results as qPCR quantification, although the PMA-qPCR treatment seemed to a good alternative to distinguish between viable and dead cells. It can be concluded that the optimized PMA-qPCR assay can be used by the industry in microbial sampling programs, helping them with the implementation of Good Agricultural Practices (GAP)Authors are thankful for the financial support from the Center for Produce Safety (Grant Agreement 2015-374) and the MINECO (Grant Agreement AGL2013-48529-R). Support provided by the COST ACTION FA1202 BacFoodNet through the concession of a Short Term Scientific Mission aimed to promote collaboration between the two research institutions is also appreciatedPeer reviewedElsevierCenter for Produce Safety (US)Ministerio de Economía y Competitividad (España)European Cooperation in Science and TechnologyAllende, Ana [0000-0002-5622-4332]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242016info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/343181reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO//AGL2013-48529-Rhttps://doi.org/10.1016/j.foodcont.2015.10.014Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3431812026-05-22T06:33:51Z
dc.title.none.fl_str_mv Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
title Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
spellingShingle Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
Truchado, Pilar
Viable cells
Microbial sampling program
Indicator microorganisms
Cultivation-based techniques
Propidium monoazide
title_short Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
title_full Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
title_fullStr Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
title_full_unstemmed Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
title_sort Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production
dc.creator.none.fl_str_mv Truchado, Pilar
Gil, Maria Isabel
Kostic, Tanja
Allende, Ana
author Truchado, Pilar
author_facet Truchado, Pilar
Gil, Maria Isabel
Kostic, Tanja
Allende, Ana
author_role author
author2 Gil, Maria Isabel
Kostic, Tanja
Allende, Ana
author2_role author
author
author
dc.contributor.none.fl_str_mv Center for Produce Safety (US)
Ministerio de Economía y Competitividad (España)
European Cooperation in Science and Technology
Allende, Ana [0000-0002-5622-4332]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Viable cells
Microbial sampling program
Indicator microorganisms
Cultivation-based techniques
Propidium monoazide
topic Viable cells
Microbial sampling program
Indicator microorganisms
Cultivation-based techniques
Propidium monoazide
description The microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in groundwater samples. This study focuses on the optimization, validation and application of a qPCR method combined with propidium monoazide (PMA) treatment to exclude DNA from dead cells. A first screening consisting of six primer sets targeting single and multi-copy of E. coli were tested to evaluate the sensitivity of the assay. After that, four primer sets were selected, combined with PMA treatment and their capacity to distinguish viable cells when combined with a background of dead cells was assessed. A primer set targeting the 23S rRNA gene was 10-fold more sensitive than the rest of primers, enabling the detection of low concentrations of viable E. coli cells. This assay also exhibited good repeatability and reproducibility, which indicates the robustness of the method. Optimized and validated PMA-qPCR was used to enumerate E. coli in environmental samples including irrigation water and fresh produce. The results were compared with the levels quantified using qPCR and cultivation-based techniques. Counts of E. coli using plate count assay were significantly lower than the levels obtained by molecular techniques (PMA-qPCR and qPCR) in both irrigation water and fresh produce. E. coli PMA-qPCR enumeration method showed similar results as qPCR quantification, although the PMA-qPCR treatment seemed to a good alternative to distinguish between viable and dead cells. It can be concluded that the optimized PMA-qPCR assay can be used by the industry in microbial sampling programs, helping them with the implementation of Good Agricultural Practices (GAP)
publishDate 2016
dc.date.none.fl_str_mv 2016
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/343181
url http://hdl.handle.net/10261/343181
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO//AGL2013-48529-R
https://doi.org/10.1016/j.foodcont.2015.10.014
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869409926847135744
score 15,811543

Similares en LA Referencia