Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples

12 pages, 4 tables, 7 figures

Detalles Bibliográficos
Autores: Rocha Grandal, David, López Cabo, Marta, Garrido-Maestu, Alejandro
Tipo de recurso: artículo
Fecha de publicación:2024
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/370894
Acceso en línea:http://hdl.handle.net/10261/370894
Access Level:acceso abierto
Palabra clave:Vibrio parahaemolyticus
Loop-mediated isothermal amplification
Internal amplification control
Reaction inhibition
Colorimetric
Mussel
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spelling Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samplesRocha Grandal, DavidLópez Cabo, MartaGarrido-Maestu, AlejandroVibrio parahaemolyticusLoop-mediated isothermal amplificationInternal amplification controlReaction inhibitionColorimetricMussel12 pages, 4 tables, 7 figuresVibrio parahaemolyticus remains as one of the most important foodborne pathogens worldwide, causing gastrointestinal disease in humans due to the consumption of raw, or undercooked, seafood. Even though molecular techniques, like PCR and qPCR, have allowed to overcome most of the limitations associated to culture-based methods, some persist such as the need for highly trained personnel, or the need to acquire expensive equipment. Loop-mediated isothermal amplification, LAMP, has emerged as a promising tool to solve these issues. The present study reports the development of a novel LAMP-based method for the detection of V. parahaemolyticus in mussels. This assay targets the gene toxR, and implements a novel, universal, freely available Internal Amplification Control (IAC) which was detected in a multiplex real-time LAMP format, where it is differentiated by melt curve analysis (86.70 ± 0.32 °C and 90.24 ± 0.34 °C for toxR and IAC respectively) and did not impact the analytical sensitivity of the target. Alternatively, the IAC was also applied in a parallel reaction and the assays performed by end-point, colorimetric LAMP. This novel IAC allowed to identify reaction inhibition due to matrix effect, and rule out false negative results. This new assay was combined with the ISO 21872 enrichment protocol, and the evaluation of spiked mussel samples indicated that an LOD95 of 14.5 and 10.8 CFU/25g could be reached after the primary 6h and secondary 24h enrichment steps by real-time LAMP, and 27.9 and 10.9 CFU/25g by naked-eye, color LAMP. It was important to note that in addition to these differences in terms of LOD95, a higher number of samples presenting reaction inhibition was observed after the primary enrichment, compared to the secondary, all successfully identified thanks to the new IAC; and also that statistically longer amplification times were required (Cq values of 19.47 ± 7.87 and 7.90 ± 1.20 respectively). The overall evaluation of the method returned values of diagnostic sensitivity, specificity and accuracy of 100 % along with an index of concordance of 1.00. These results were consistent regardless if the real-time, or the colorimetric, LAMP strategy was followedThe Marine Science Program, ThinkInAzul, in Galicia supported by Ministerio de Ciencia e Innovación and Xunta de Galicia (Working Package: PT3) with funding from European Union NextGenerationEU (PRTR-C17.I1) and European Maritime and Fisheries FundPeer reviewedElsevierMinisterio de Ciencia e Innovación (España)Xunta de GaliciaEuropean CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242025info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/370894reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésPostprintThe underlying dataset has been published as supplementary material of the article in the publishers platform at DOI https://doi.org/10.1016/j.foodcont.2024.110965https://doi.org/10.1016/j.foodcont.2024.110965Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3708942026-05-22T06:33:51Z
dc.title.none.fl_str_mv Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
title Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
spellingShingle Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
Rocha Grandal, David
Vibrio parahaemolyticus
Loop-mediated isothermal amplification
Internal amplification control
Reaction inhibition
Colorimetric
Mussel
title_short Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
title_full Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
title_fullStr Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
title_full_unstemmed Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
title_sort Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples
dc.creator.none.fl_str_mv Rocha Grandal, David
López Cabo, Marta
Garrido-Maestu, Alejandro
author Rocha Grandal, David
author_facet Rocha Grandal, David
López Cabo, Marta
Garrido-Maestu, Alejandro
author_role author
author2 López Cabo, Marta
Garrido-Maestu, Alejandro
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Xunta de Galicia
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Vibrio parahaemolyticus
Loop-mediated isothermal amplification
Internal amplification control
Reaction inhibition
Colorimetric
Mussel
topic Vibrio parahaemolyticus
Loop-mediated isothermal amplification
Internal amplification control
Reaction inhibition
Colorimetric
Mussel
description 12 pages, 4 tables, 7 figures
publishDate 2024
dc.date.none.fl_str_mv 2024
2024
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/370894
url http://hdl.handle.net/10261/370894
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Postprint
The underlying dataset has been published as supplementary material of the article in the publishers platform at DOI https://doi.org/10.1016/j.foodcont.2024.110965
https://doi.org/10.1016/j.foodcont.2024.110965
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
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score 15,811543

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