Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes.
Liou, Roxanne Hsiang-Chi
Hung, Sandy Shen-Chi
Hewitt, Alex W
Martin, Keith R
Edwards, Thomas L
Wong, Raymond Ching-Bong
Springer Science and Business Media LLC
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Urrutia-Cabrera, D., Liou, R. H., Wang, J., Chan, J., Hung, S. S., Hewitt, A. W., Martin, K. R., et al. (2021). Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes.. Sci Rep, 11 (1. 22493), 22493. https://doi.org/10.1038/s41598-021-01472-3
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.
Adult, COVID-19, COVID-19 Nucleic Acid Testing, Female, Humans, Male, Molecular Diagnostic Techniques, Nucleic Acid Amplification Techniques, SARS-CoV-2, Saliva
External DOI: https://doi.org/10.1038/s41598-021-01472-3
This record's URL: https://www.repository.cam.ac.uk/handle/1810/332864
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/