Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat

Autores
Brusa, Victoria; Galli, Lucía; Linares, Luciano H.; Ortega, Emanuel E.; Liron, Juan Pedro; Leotta, Gerardo Anibal
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa = 0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.
Fil: Brusa, Victoria. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; Argentina
Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
Fil: Linares, Luciano H.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina
Fil: Ortega, Emanuel E.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina
Fil: Liron, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
Fil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
Materia
Shiga Toxin-Producing Escherichia Coli
Sybr-Pcr
Rt-Pcr
Ground Beef
Stx Genes
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/11506
Acceder
id CONICETDig_41065e7c5360baba8594340bc1db7d57
oai_identifier_str oai:ri.conicet.gov.ar:11336/11506
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meatBrusa, VictoriaGalli, LucíaLinares, Luciano H.Ortega, Emanuel E.Liron, Juan PedroLeotta, Gerardo AnibalShiga Toxin-Producing Escherichia ColiSybr-PcrRt-PcrGround BeefStx Geneshttps://purl.org/becyt/ford/4.3https://purl.org/becyt/ford/4Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa = 0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.Fil: Brusa, Victoria. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; ArgentinaFil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; ArgentinaFil: Linares, Luciano H.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; ArgentinaFil: Ortega, Emanuel E.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; ArgentinaFil: Liron, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; ArgentinaFil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; ArgentinaElsevier Science2015-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/11506Brusa, Victoria; Galli, Lucía; Linares, Luciano H.; Ortega, Emanuel E.; Liron, Juan Pedro; et al.; Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat; Elsevier Science; Journal Of Microbiological Methods; 119; 12-2015; 10-170167-7012enginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.mimet.2015.09.013info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0167701215300695info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:53:48Zoai:ri.conicet.gov.ar:11336/11506instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:53:48.347CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
spellingShingle Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
Brusa, Victoria
Shiga Toxin-Producing Escherichia Coli
Sybr-Pcr
Rt-Pcr
Ground Beef
Stx Genes
title_short Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_full Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_fullStr Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_full_unstemmed Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
title_sort Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat
dc.creator.none.fl_str_mv Brusa, Victoria
Galli, Lucía
Linares, Luciano H.
Ortega, Emanuel E.
Liron, Juan Pedro
Leotta, Gerardo Anibal
author Brusa, Victoria
author_facet Brusa, Victoria
Galli, Lucía
Linares, Luciano H.
Ortega, Emanuel E.
Liron, Juan Pedro
Leotta, Gerardo Anibal
author_role author
author2 Galli, Lucía
Linares, Luciano H.
Ortega, Emanuel E.
Liron, Juan Pedro
Leotta, Gerardo Anibal
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Shiga Toxin-Producing Escherichia Coli
Sybr-Pcr
Rt-Pcr
Ground Beef
Stx Genes
topic Shiga Toxin-Producing Escherichia Coli
Sybr-Pcr
Rt-Pcr
Ground Beef
Stx Genes
purl_subject.fl_str_mv https://purl.org/becyt/ford/4.3
https://purl.org/becyt/ford/4
dc.description.none.fl_txt_mv Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa = 0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.
Fil: Brusa, Victoria. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Genética Veterinaria "Ingeniero Fernando Noel Dulout"; Argentina
Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
Fil: Linares, Luciano H.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina
Fil: Ortega, Emanuel E.. Universidad Nacional de la Plata. Facultad de Cs.veterinarias. Departamento de Epizootiologia y Salud Publica. Laboratorio de Microbiologia de Alimentos; Argentina
Fil: Liron, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
Fil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Genética Veterinaria "ingeniero Fernando Noel Dulout"; Argentina
description Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n = 103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stxpositive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1 × 102 CFU mL−1 limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa = 0.758 and 0.801 (P b 0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.
publishDate 2015
dc.date.none.fl_str_mv 2015-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/11506
Brusa, Victoria; Galli, Lucía; Linares, Luciano H.; Ortega, Emanuel E.; Liron, Juan Pedro; et al.; Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat; Elsevier Science; Journal Of Microbiological Methods; 119; 12-2015; 10-17
0167-7012
url http://hdl.handle.net/11336/11506
identifier_str_mv Brusa, Victoria; Galli, Lucía; Linares, Luciano H.; Ortega, Emanuel E.; Liron, Juan Pedro; et al.; Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat; Elsevier Science; Journal Of Microbiological Methods; 119; 12-2015; 10-17
0167-7012
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.mimet.2015.09.013
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0167701215300695
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.13397

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