Abstract
The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.
Highlights
The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus [1], is a major global health threat
Compared to an reverse transcription (RT)-qPCR assay using a sensitive primer set, we found that the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%
Several primer sets were recently proposed for RT-LAMPbased detection of SARS-CoV-2 RNA by Zhang et al [11] as well as by Yu et al [10], and these primer sets were subsequently validated with in vitro translated RNA
Summary
The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus [1], is a major global health threat. Due to limited testing capacity, only people with symptoms are usually tested for SARS-CoV-2 infection, studies have confirmed that many individuals infected with SARS-CoV-2 are asymptomatic carriers of the virus [3, 4]. This suggests that infectioncontrol strategies focusing on symptomatic patients are not sufficient to prevent virus spread. Large scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively This would allow for the rapid isolation of infected persons during an existing wave of infection and to identify newly emerging outbreaks. Continuous and repeated testing of large groups within a population may be required as a long-term strategy to contain new outbreaks while keeping societies and economies functional until effective vaccines become available
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