Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples
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). Bo...
| Autores principales: | , , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
| Publicado: |
Springer
2024
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| Acceso en línea: | 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 |
| _version_ | 1855486139276722176 |
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| author | Zbrun, María Virginia Moreno, Nadia Camussone, Cecilia Signorini Porchiett, Marcelo Lisandro Primo, María Evangelina |
| author_browse | Camussone, Cecilia Moreno, Nadia Primo, María Evangelina Signorini Porchiett, Marcelo Lisandro Zbrun, María Virginia |
| author_facet | Zbrun, María Virginia Moreno, Nadia Camussone, Cecilia Signorini Porchiett, Marcelo Lisandro Primo, María Evangelina |
| author_sort | Zbrun, María Virginia |
| collection | INTA Digital |
| 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. |
| format | Artículo |
| id | INTA18314 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Springer |
| publisherStr | Springer |
| record_format | dspace |
| spelling | INTA183142025-09-12T11:54:41Z 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 Moreno, Nadia Camussone, Cecilia Signorini Porchiett, Marcelo Lisandro Primo, María Evangelina Listeria monocytogenes Cheese PCR Soft Cheese Foods Queso Queso Blando Alimentos 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 2024-06-28T14:56:30Z 2024-06-28T14:56:30Z 2024-04 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng 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) application/pdf Springer Brazilian Journal of Microbiology 55 : 1783-1791. (April 2024) |
| spellingShingle | Listeria monocytogenes Cheese PCR Soft Cheese Foods Queso Queso Blando Alimentos Zbrun, María Virginia Moreno, Nadia Camussone, Cecilia Signorini Porchiett, Marcelo Lisandro Primo, María Evangelina 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 |
| 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_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_sort | comparison of real time pcr and nested pcr based on the hlya gene for the detection of listeria monocytogenes application on cheese samples |
| topic | Listeria monocytogenes Cheese PCR Soft Cheese Foods Queso Queso Blando Alimentos |
| 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 |
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