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

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 pa...

Full description

Bibliographic Details
Main Authors: Eduardo Juscamayta-López, Faviola Valdivia, Helen Horna, David Tarazona, Liza Linares, Nancy Rojas, Maribel Huaringa
Format: Article
Language:English
Published: Frontiers Media S.A. 2021-06-01
Series:Frontiers in Cellular and Infection Microbiology
Subjects:
Online Access:https://www.frontiersin.org/articles/10.3389/fcimb.2021.653616/full
_version_ 1818739024752279552
author Eduardo Juscamayta-López
Faviola Valdivia
Helen Horna
David Tarazona
Liza Linares
Nancy Rojas
Maribel Huaringa
author_facet Eduardo Juscamayta-López
Faviola Valdivia
Helen Horna
David Tarazona
Liza Linares
Nancy Rojas
Maribel Huaringa
author_sort Eduardo Juscamayta-López
collection DOAJ
description 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.
first_indexed 2024-12-18T01:18:16Z
format Article
id doaj.art-69368a23e8e845c38f7b38215f7e9f00
institution Directory Open Access Journal
issn 2235-2988
language English
last_indexed 2024-12-18T01:18:16Z
publishDate 2021-06-01
publisher Frontiers Media S.A.
record_format Article
series Frontiers in Cellular and Infection Microbiology
spelling doaj.art-69368a23e8e845c38f7b38215f7e9f002022-12-21T21:25:54ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882021-06-011110.3389/fcimb.2021.653616653616A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2Eduardo Juscamayta-López0Faviola Valdivia1Helen Horna2David Tarazona3Liza Linares4Nancy Rojas5Maribel Huaringa6Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Virus Respiratorios, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruLaboratorio de Virus Respiratorios, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, PeruCoronavirus 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.https://www.frontiersin.org/articles/10.3389/fcimb.2021.653616/fullMultiplex RT-LAMPSARS-CoV-2COVID-19rapid molecular teststimely diagnosisdiagnostic accuracy
spellingShingle Eduardo Juscamayta-López
Faviola Valdivia
Helen Horna
David Tarazona
Liza Linares
Nancy Rojas
Maribel Huaringa
A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
Frontiers in Cellular and Infection Microbiology
Multiplex RT-LAMP
SARS-CoV-2
COVID-19
rapid molecular tests
timely diagnosis
diagnostic accuracy
title 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_short A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
title_sort multiplex and colorimetric reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of novel sars cov 2
topic Multiplex RT-LAMP
SARS-CoV-2
COVID-19
rapid molecular tests
timely diagnosis
diagnostic accuracy
url https://www.frontiersin.org/articles/10.3389/fcimb.2021.653616/full
work_keys_str_mv AT eduardojuscamaytalopez amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT faviolavaldivia amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT helenhorna amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT davidtarazona amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT lizalinares amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT nancyrojas amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT maribelhuaringa amultiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT eduardojuscamaytalopez multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT faviolavaldivia multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT helenhorna multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT davidtarazona multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT lizalinares multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT nancyrojas multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2
AT maribelhuaringa multiplexandcolorimetricreversetranscriptionloopmediatedisothermalamplificationassayforsensitiveandrapiddetectionofnovelsarscov2