High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Sanchita Bhadra; Timothy E Riedel; Simren Lakhotia; Nicholas D Tran; Andrew D Ellington

Integration of OSD probes into pre-published SARS-CoV-2 LAMP primer sets A series of 11 published primer sets for SARS-CoV-2 were screened using New England Biolab's WarmStart® Colorimetric LAMP 2X Master Mix (NEB, Ipswich, MA, USA) according to the manufacturer's protocol ( Table 2) . We found that 9 of the 11 sets showed significant no-template amplification in over 10% of the replicates in less than an hour of incubation at 65 °C (data not shown). These results are consistent with other published results that rely on colorimetric LAMP reactions, rather than on probe-based detection. 21 In fact, for many published assays, color changes must be read within a narrow window of time in order to minimize spurious conclusions, a consideration that does not scale well for diagnostic screening, especially at point-of-care or as an early part of a clinical diagnostics pipeline. To suppress potential false positive readout, we chose to develop OSD probes for three of the LAMP primer sets, termed herein as NB, Lamb, and Tholoth ( Table 1) . These primer sets target three different regions of the viral genome, the N gene, the NSP3 coding region of ORF1AB, and the RNA-dependent RNA polymerase coding region of ORF1AB. Of the three primer sets, the NB assay had the lowest propensity for spurious signal when analyzed by non-specific colorimetric readout (data not shown). To create LAMP-OSD versions of these assays, we designed OSD probes that were complementary to one of the loop sequences in each of the three LAMP amplicons. Subsequently, Tholoth, Lamb, and NB LAMP-OSD assays were setup individually by mixing separate reaction components as indicated in the Methods section. Each individual assay contained its specific OSD probes along with both inner primers FIP and BIP and both outer primers F3 and B3. In addition, each assay also received the backward loop (LB) primer that bound to the amplicon loop between B1c and B2 sites that was not recognized by the respective OSD probe. The forward loop (LF) primer that overlapped the Tholoth and NB OSD binding region was excluded. The LF primer was also initially excluded in Lamb LAMP-OSD assay even though the amplicon loop that bound this loop primer was long enough to accommodate a nonoverlapping OSD reporter; this was done to fairly compare the amplification kinetics of all three assays in a 5-primer format. In later versions of the assay with the Lamb primers, all 6 primers were included (designated as "6 primer Lamb" in Figures 3, 6, and 7) . Figure 2 , in response to target templates, all three LAMP-OSD assays generated strong OSD signal that could be measured both in real-time and observed visually at endpoint without interference from noise. We then tested the three LAMP-OSD assays using SARS-CoV-2 genomic RNA as templates.