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

Abstract 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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Daniel Urrutia-Cabrera, Roxanne Hsiang-Chi Liou, Jiang-Hui Wang, Jianxiong Chan, Sandy Shen-Chi Hung, Alex W. Hewitt, Keith R. Martin, Thomas L. Edwards, Patrick Kwan, Raymond Ching-Bong Wong
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/cafa6f5cd3294e629d44f28865896d6b
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:cafa6f5cd3294e629d44f28865896d6b
record_format	dspace
spelling	oai:doaj.org-article:cafa6f5cd3294e629d44f28865896d6b2021-11-21T12:16:20ZComparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes10.1038/s41598-021-01472-32045-2322https://doaj.org/article/cafa6f5cd3294e629d44f28865896d6b2021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01472-3https://doaj.org/toc/2045-2322Abstract 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.Daniel Urrutia-CabreraRoxanne Hsiang-Chi LiouJiang-Hui WangJianxiong ChanSandy Shen-Chi HungAlex W. HewittKeith R. MartinThomas L. EdwardsPatrick KwanRaymond Ching-Bong WongNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniel Urrutia-Cabrera Roxanne Hsiang-Chi Liou Jiang-Hui Wang Jianxiong Chan Sandy Shen-Chi Hung Alex W. Hewitt Keith R. Martin Thomas L. Edwards Patrick Kwan Raymond Ching-Bong Wong Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes
description	Abstract 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.
format	article
author	Daniel Urrutia-Cabrera Roxanne Hsiang-Chi Liou Jiang-Hui Wang Jianxiong Chan Sandy Shen-Chi Hung Alex W. Hewitt Keith R. Martin Thomas L. Edwards Patrick Kwan Raymond Ching-Bong Wong
author_facet	Daniel Urrutia-Cabrera Roxanne Hsiang-Chi Liou Jiang-Hui Wang Jianxiong Chan Sandy Shen-Chi Hung Alex W. Hewitt Keith R. Martin Thomas L. Edwards Patrick Kwan Raymond Ching-Bong Wong
author_sort	Daniel Urrutia-Cabrera
title	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_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_sort	comparative analysis of loop-mediated isothermal amplification (lamp)-based assays for rapid detection of sars-cov-2 genes
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/cafa6f5cd3294e629d44f28865896d6b
work_keys_str_mv	AT danielurrutiacabrera comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT roxannehsiangchiliou comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT jianghuiwang comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT jianxiongchan comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT sandyshenchihung comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT alexwhewitt comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT keithrmartin comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT thomasledwards comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT patrickkwan comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes AT raymondchingbongwong comparativeanalysisofloopmediatedisothermalamplificationlampbasedassaysforrapiddetectionofsarscov2genes
_version_	1718419150357921792

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

Ejemplares similares