A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2

Descripción del Articulo

This work was supported by the CONCYTEC-FONDECYT Program of Proyectos Especiales: Respuesta al COVID-19 2020-01-01 [grant number 034-2020-FONDECYT] and the National Institute of Health of Peru.
Detalles Bibliográficos
Autores: Juscamayta-López, E., Valdivia, F., Horna, H., Tarazona, D., Linares, L., Rojas, N., Huaringa, M.
Formato: artículo
Fecha de Publicación:2021
Institución:Consejo Nacional de Ciencia Tecnología e Innovación
Repositorio:CONCYTEC-Institucional
Lenguaje:inglés
OAI Identifier:oai:repositorio.concytec.gob.pe:20.500.12390/2960
Enlace del recurso:https://hdl.handle.net/20.500.12390/2960
https://doi.org/10.3389/fcimb.2021.653616
Nivel de acceso:acceso abierto
Materia:timely diagnosis
COVID-19
diagnostic accuracy
Multiplex RT-LAMP
primary health care
rapid molecular tests
resource-limited settings
SARS-CoV-2
https://purl.org/pe-repo/ocde/ford#3.03.08
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oai_identifier_str oai:repositorio.concytec.gob.pe:20.500.12390/2960
network_acronym_str CONC
network_name_str CONCYTEC-Institucional
repository_id_str 4689
dc.title.none.fl_str_mv A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
spellingShingle A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
Juscamayta-López, E.
timely diagnosis
COVID-19
diagnostic accuracy
Multiplex RT-LAMP
Multiplex RT-LAMP
primary health care
rapid molecular tests
resource-limited settings
resource-limited settings
SARS-CoV-2
SARS-CoV-2
https://purl.org/pe-repo/ocde/ford#3.03.08
title_short A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title_full A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title_fullStr A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title_full_unstemmed A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title_sort A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
author Juscamayta-López, E.
author_facet Juscamayta-López, E.
Valdivia, F.
Horna, H.
Tarazona, D.
Linares, L.
Rojas, N.
Huaringa, M.
author_role author
author2 Valdivia, F.
Horna, H.
Tarazona, D.
Linares, L.
Rojas, N.
Huaringa, M.
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Juscamayta-López, E.
Valdivia, F.
Horna, H.
Tarazona, D.
Linares, L.
Rojas, N.
Huaringa, M.
dc.subject.none.fl_str_mv timely diagnosis
topic timely diagnosis
COVID-19
diagnostic accuracy
Multiplex RT-LAMP
Multiplex RT-LAMP
primary health care
rapid molecular tests
resource-limited settings
resource-limited settings
SARS-CoV-2
SARS-CoV-2
https://purl.org/pe-repo/ocde/ford#3.03.08
dc.subject.es_PE.fl_str_mv COVID-19
diagnostic accuracy
Multiplex RT-LAMP
Multiplex RT-LAMP
primary health care
rapid molecular tests
resource-limited settings
resource-limited settings
SARS-CoV-2
SARS-CoV-2
dc.subject.ocde.none.fl_str_mv https://purl.org/pe-repo/ocde/ford#3.03.08
description This work was supported by the CONCYTEC-FONDECYT Program of Proyectos Especiales: Respuesta al COVID-19 2020-01-01 [grant number 034-2020-FONDECYT] and the National Institute of Health of Peru.
publishDate 2021
dc.date.accessioned.none.fl_str_mv 2024-05-30T23:13:38Z
dc.date.available.none.fl_str_mv 2024-05-30T23:13:38Z
dc.date.issued.fl_str_mv 2021
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.uri.none.fl_str_mv https://hdl.handle.net/20.500.12390/2960
dc.identifier.doi.none.fl_str_mv https://doi.org/10.3389/fcimb.2021.653616
dc.identifier.scopus.none.fl_str_mv 2-s2.0-85110180024
url https://hdl.handle.net/20.500.12390/2960
https://doi.org/10.3389/fcimb.2021.653616
identifier_str_mv 2-s2.0-85110180024
dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.ispartof.none.fl_str_mv Frontiers in Cellular and Infection Microbiology
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
dc.rights.uri.none.fl_str_mv https://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/4.0/
dc.publisher.none.fl_str_mv Frontiers Media S.A.
publisher.none.fl_str_mv Frontiers Media S.A.
dc.source.none.fl_str_mv reponame:CONCYTEC-Institucional
instname:Consejo Nacional de Ciencia Tecnología e Innovación
instacron:CONCYTEC
instname_str Consejo Nacional de Ciencia Tecnología e Innovación
instacron_str CONCYTEC
institution CONCYTEC
reponame_str CONCYTEC-Institucional
collection CONCYTEC-Institucional
repository.name.fl_str_mv Repositorio Institucional CONCYTEC
repository.mail.fl_str_mv repositorio@concytec.gob.pe
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spelling Publicationrp08383600rp08382600rp08380600rp08379600rp08384600rp06429600rp08381600Juscamayta-López, E.Valdivia, F.Horna, H.Tarazona, D.Linares, L.Rojas, N.Huaringa, M.2024-05-30T23:13:38Z2024-05-30T23:13:38Z2021https://hdl.handle.net/20.500.12390/2960https://doi.org/10.3389/fcimb.2021.6536162-s2.0-85110180024This work was supported by the CONCYTEC-FONDECYT Program of Proyectos Especiales: Respuesta al COVID-19 2020-01-01 [grant number 034-2020-FONDECYT] and the National Institute of Health of Peru.Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device. © Copyright © 2021 Juscamayta-López, Valdivia, Horna, Tarazona, Linares, Rojas and Huaringa.Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica - ConcytecengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiologyinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/4.0/timely diagnosisCOVID-19-1diagnostic accuracy-1Multiplex RT-LAMP-1Multiplex RT-LAMP-1primary health care-1rapid molecular tests-1resource-limited settings-1resource-limited settings-1SARS-CoV-2-1SARS-CoV-2-1https://purl.org/pe-repo/ocde/ford#3.03.08-1A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2info:eu-repo/semantics/articlereponame:CONCYTEC-Institucionalinstname:Consejo Nacional de Ciencia Tecnología e Innovacióninstacron:CONCYTEC20.500.12390/2960oai:repositorio.concytec.gob.pe:20.500.12390/29602024-05-30 16:12:30.846https://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_14cbinfo:eu-repo/semantics/closedAccessmetadata only accesshttps://repositorio.concytec.gob.peRepositorio Institucional CONCYTECrepositorio@concytec.gob.pe#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#<Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="7cf9cf30-4af2-423e-a2ef-2734b1417e27"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2</Title> <PublishedIn> <Publication> <Title>Frontiers in Cellular and Infection Microbiology</Title> </Publication> </PublishedIn> <PublicationDate>2021</PublicationDate> <DOI>https://doi.org/10.3389/fcimb.2021.653616</DOI> <SCP-Number>2-s2.0-85110180024</SCP-Number> <Authors> <Author> <DisplayName>Juscamayta-López, E.</DisplayName> <Person id="rp08383" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Valdivia, F.</DisplayName> <Person id="rp08382" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Horna, H.</DisplayName> <Person id="rp08380" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Tarazona, D.</DisplayName> <Person id="rp08379" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Linares, L.</DisplayName> <Person id="rp08384" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Rojas, N.</DisplayName> <Person id="rp06429" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> <Author> <DisplayName>Huaringa, M.</DisplayName> <Person id="rp08381" /> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Frontiers Media S.A.</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>https://creativecommons.org/licenses/by-nc-nd/4.0/</License> <Keyword>timely diagnosis</Keyword> <Keyword>COVID-19</Keyword> <Keyword>diagnostic accuracy</Keyword> <Keyword>Multiplex RT-LAMP</Keyword> <Keyword>Multiplex RT-LAMP</Keyword> <Keyword>primary health care</Keyword> <Keyword>rapid molecular tests</Keyword> <Keyword>resource-limited settings</Keyword> <Keyword>resource-limited settings</Keyword> <Keyword>SARS-CoV-2</Keyword> <Keyword>SARS-CoV-2</Keyword> <Abstract>Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p&lt;0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (&lt;45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device. © Copyright © 2021 Juscamayta-López, Valdivia, Horna, Tarazona, Linares, Rojas and Huaringa.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication> -1
score 13.392684
A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
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