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Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes

The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnosti...

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Autores principales: Urrutia-Cabrera, Daniel, Liou, Roxanne Hsiang-Chi, Wang, Jiang-Hui, Chan, Jianxiong, Hung, Sandy Shen-Chi, Hewitt, Alex W., Martin, Keith R., Edwards, Thomas L., Kwan, Patrick, Wong, Raymond Ching-Bong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602269/
https://www.ncbi.nlm.nih.gov/pubmed/34795308
http://dx.doi.org/10.1038/s41598-021-01472-3
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author Urrutia-Cabrera, Daniel
Liou, Roxanne Hsiang-Chi
Wang, Jiang-Hui
Chan, Jianxiong
Hung, Sandy Shen-Chi
Hewitt, Alex W.
Martin, Keith R.
Edwards, Thomas L.
Kwan, Patrick
Wong, Raymond Ching-Bong
author_facet Urrutia-Cabrera, Daniel
Liou, Roxanne Hsiang-Chi
Wang, Jiang-Hui
Chan, Jianxiong
Hung, Sandy Shen-Chi
Hewitt, Alex W.
Martin, Keith R.
Edwards, Thomas L.
Kwan, Patrick
Wong, Raymond Ching-Bong
author_sort Urrutia-Cabrera, Daniel
collection PubMed
description The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.
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spelling pubmed-86022692021-11-19 Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes Urrutia-Cabrera, Daniel Liou, Roxanne Hsiang-Chi Wang, Jiang-Hui Chan, Jianxiong Hung, Sandy Shen-Chi Hewitt, Alex W. Martin, Keith R. Edwards, Thomas L. Kwan, Patrick Wong, Raymond Ching-Bong Sci Rep Article The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2. Nature Publishing Group UK 2021-11-18 /pmc/articles/PMC8602269/ /pubmed/34795308 http://dx.doi.org/10.1038/s41598-021-01472-3 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Urrutia-Cabrera, Daniel
Liou, Roxanne Hsiang-Chi
Wang, Jiang-Hui
Chan, Jianxiong
Hung, Sandy Shen-Chi
Hewitt, Alex W.
Martin, Keith R.
Edwards, Thomas L.
Kwan, Patrick
Wong, Raymond Ching-Bong
Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title_full Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title_fullStr Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title_full_unstemmed Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title_short Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
title_sort comparative analysis of loop-mediated isothermal amplification (lamp)-based assays for rapid detection of sars-cov-2 genes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602269/
https://www.ncbi.nlm.nih.gov/pubmed/34795308
http://dx.doi.org/10.1038/s41598-021-01472-3
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