A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

Abstract

The COVID-19 pandemic caused by the SARS-CoV-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-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.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

“…These results are well in agreement with the recently reported sensitivity cutoff at CT 30 for the RT-LAMP assay [7] and, thus, confirm that RNA purified by the presented magnetic bead extraction is compatible with RT-LAMP detection without lowering its sensitivity.…”

“…RT-LAMP detection of the SARS-CoV-2 N gene was based on the protocol by Dao Thi et al [7]. The RT-LAMP primer set used in this study was targeted against the SARS-CoV-2 N gene [17].…”

“…The RT-LAMP primer set used in this study was targeted against the SARS-CoV-2 N gene [17]. The sequences and concentrations of all oligonucleotides in the 10x primer mix used for the RT-LAMP assays can be found in our recent publication [7]. For the colorimetric LAMP assay, the master mix consisted of 6.25 µl WarmStart ® Colorimetric LAMP 2X Master Mix M1800 (New England Biolabs, Ipswich, MA, USA) and 1.25 µl 10× LAMP primer mix targeting the N gene [17] per reaction.…”

“…We also recently explored the RT-LAMP assay as a valid alternative RNA detection method due to shortages in RT-qPCR reagents [7]. We compared the standard pipeline QIAcube RNA extraction followed by RT-qPCR detection of the E gene with magnetic RNA extraction followed by RT-LAMP detection using primer sets targeting the N gene, with either colorimetric ( Figure 5A-C, Supplementary Table S2) or fluorescent ( Figure 5D-F, Supplementary Table S3) read-out.…”

“…A two-step testing procedure using primer sets targeting the E gene for initial screening followed by the RdRp gene to confirm positive samples is recommended by the German Consiliary Laboratory for Coronaviruses [2]. The unprecedented global demand for commercial RNA extraction kits and ensuing shortage of these reagents [3] led to the establishment of several diagnostic workflows performed on patient samples with or without an intermediate RNA extraction step [4][5][6][7]. Viral RNA isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization, and on the denaturation of omnipresent RNases [8].…”