Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay

In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and manageme...

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Autores: Montagud-Martínez, R, Marquez-Costa, R, Ruiz, R, Martínez-Aviñó, A, Ballesteros-Garrido, R, Navarro, D, Campins-Falcó, P, Rodrigo, G
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Institución:INCLIVA
Repositorio:r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVA
OAI Identifier:oai:incliva.fundanetsuite.com:p19960
Acceso en línea:https://incliva.portalinvestigacion.com/publicaciones/19960
Access Level:acceso abierto
Palabra clave:bioanalytics
CRISPR diagnostics
DNA nanotechnology
lateral flow immunochromatography
synthetic biology
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spelling Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow AssayMontagud-Martínez, RMarquez-Costa, RRuiz, RMartínez-Aviñó, ABallesteros-Garrido, RNavarro, DCampins-Falcó, PRodrigo, GbioanalyticsCRISPR diagnosticsDNA nanotechnologylateral flow immunochromatographysynthetic biologyIn public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labeled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/mu L in 1 h at a low temperature. We validated the efficacy of the method by using clinical samples of patients infected with SARS-CoV-2. Compared with other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving the simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.AMER CHEMICAL SOC2025info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://incliva.portalinvestigacion.com/publicaciones/19960ACS Applied Bio MaterialsISSN: 25766422reponame:r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVAinstname:INCLIVAInglésinfo:eu-repo/semantics/openAccessoai:incliva.fundanetsuite.com:p199602026-06-07T16:35:31Z
dc.title.none.fl_str_mv Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
title Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
spellingShingle Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
Montagud-Martínez, R
bioanalytics
CRISPR diagnostics
DNA nanotechnology
lateral flow immunochromatography
synthetic biology
title_short Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
title_full Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
title_fullStr Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
title_full_unstemmed Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
title_sort Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay
dc.creator.none.fl_str_mv Montagud-Martínez, R
Marquez-Costa, R
Ruiz, R
Martínez-Aviñó, A
Ballesteros-Garrido, R
Navarro, D
Campins-Falcó, P
Rodrigo, G
author Montagud-Martínez, R
author_facet Montagud-Martínez, R
Marquez-Costa, R
Ruiz, R
Martínez-Aviñó, A
Ballesteros-Garrido, R
Navarro, D
Campins-Falcó, P
Rodrigo, G
author_role author
author2 Marquez-Costa, R
Ruiz, R
Martínez-Aviñó, A
Ballesteros-Garrido, R
Navarro, D
Campins-Falcó, P
Rodrigo, G
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv bioanalytics
CRISPR diagnostics
DNA nanotechnology
lateral flow immunochromatography
synthetic biology
topic bioanalytics
CRISPR diagnostics
DNA nanotechnology
lateral flow immunochromatography
synthetic biology
description In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labeled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/mu L in 1 h at a low temperature. We validated the efficacy of the method by using clinical samples of patients infected with SARS-CoV-2. Compared with other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving the simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.
publishDate 2025
dc.date.none.fl_str_mv 2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://incliva.portalinvestigacion.com/publicaciones/19960
url https://incliva.portalinvestigacion.com/publicaciones/19960
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv AMER CHEMICAL SOC
publisher.none.fl_str_mv AMER CHEMICAL SOC
dc.source.none.fl_str_mv ACS Applied Bio Materials
ISSN: 25766422
reponame:r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVA
instname:INCLIVA
instname_str INCLIVA
reponame_str r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVA
collection r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVA
repository.name.fl_str_mv
repository.mail.fl_str_mv
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