Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples

Autores
Zbrun, María Virginia; Moreno, Nadia; Camussone, Cecilia; Signorini Porchiett, Marcelo Lisandro; Primo, María Evangelina
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.
EEA Rafaela
Fil: Zbrun, Maria Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Zbrun, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Zbrun, Maria Virginia. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; Argentina
Fil: Moreno, Nadia. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; Argentina
Fil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Camussone, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; Argentina
Fil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; Argentina
Fuente
Brazilian Journal of Microbiology 55 : 1783-1791. (April 2024)
Materia
Listeria monocytogenes
Cheese
PCR
Soft Cheese
Foods
Queso
Queso Blando
Alimentos
Nivel de accesibilidad
acceso restringido
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/18314
Acceder
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oai_identifier_str oai:localhost:20.500.12123/18314
network_acronym_str INTADig
repository_id_str l
network_name_str INTA Digital (INTA)
spelling Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samplesZbrun, María VirginiaMoreno, NadiaCamussone, CeciliaSignorini Porchiett, Marcelo LisandroPrimo, María EvangelinaListeria monocytogenesCheesePCRSoft CheeseFoodsQuesoQueso BlandoAlimentosThe aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.EEA RafaelaFil: Zbrun, Maria Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Zbrun, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Zbrun, Maria Virginia. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; ArgentinaFil: Moreno, Nadia. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; ArgentinaFil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Camussone, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Signorini, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Signorini, Marcelo. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; ArgentinaFil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Primo, María Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Primo, María Evangelina. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; ArgentinaSpringer2024-06-28T14:56:30Z2024-06-28T14:56:30Z2024-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/18314https://link.springer.com/article/10.1007/s42770-024-01353-71517-83821678-4405https://doi.org/10.1007/s42770-024-01353-7Brazilian Journal of Microbiology 55 : 1783-1791. (April 2024)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-18T10:09:30Zoai:localhost:20.500.12123/18314instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-18 10:09:31.371INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
title Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
spellingShingle Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
Zbrun, María Virginia
Listeria monocytogenes
Cheese
PCR
Soft Cheese
Foods
Queso
Queso Blando
Alimentos
title_short Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
title_full Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
title_fullStr Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
title_full_unstemmed Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
title_sort Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
dc.creator.none.fl_str_mv Zbrun, María Virginia
Moreno, Nadia
Camussone, Cecilia
Signorini Porchiett, Marcelo Lisandro
Primo, María Evangelina
author Zbrun, María Virginia
author_facet Zbrun, María Virginia
Moreno, Nadia
Camussone, Cecilia
Signorini Porchiett, Marcelo Lisandro
Primo, María Evangelina
author_role author
author2 Moreno, Nadia
Camussone, Cecilia
Signorini Porchiett, Marcelo Lisandro
Primo, María Evangelina
author2_role author
author
author
author
dc.subject.none.fl_str_mv Listeria monocytogenes
Cheese
PCR
Soft Cheese
Foods
Queso
Queso Blando
Alimentos
topic Listeria monocytogenes
Cheese
PCR
Soft Cheese
Foods
Queso
Queso Blando
Alimentos
dc.description.none.fl_txt_mv The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.
EEA Rafaela
Fil: Zbrun, Maria Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Zbrun, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Zbrun, Maria Virginia. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; Argentina
Fil: Moreno, Nadia. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; Argentina
Fil: Camussone, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Camussone, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Signorini, Marcelo. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Departamento de Salud Pública; Argentina
Fil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Universidad Nacional de Rafaela. Facultad de Tecnologías e Innovación para el Desarrollo; Argentina
description The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290–1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290–1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.
publishDate 2024
dc.date.none.fl_str_mv 2024-06-28T14:56:30Z
2024-06-28T14:56:30Z
2024-04
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/20.500.12123/18314
https://link.springer.com/article/10.1007/s42770-024-01353-7
1517-8382
1678-4405
https://doi.org/10.1007/s42770-024-01353-7
url http://hdl.handle.net/20.500.12123/18314
https://link.springer.com/article/10.1007/s42770-024-01353-7
https://doi.org/10.1007/s42770-024-01353-7
identifier_str_mv 1517-8382
1678-4405
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv restrictedAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv Brazilian Journal of Microbiology 55 : 1783-1791. (April 2024)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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