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Design and Selection of RT-LAMP Primer Sets Targeting SARS-CoV-2 in Complex Human Samples
Loop-mediated Isothermal Amplification (LAMP) is a promising technology to address diagnostic and surveillance testing during public health crises, such as the current COVID-19 pandemic; however, primer design and assay optimization remain a barrier to enabling rapid deployment of assays based on LAMP. Herein, we introduce a design and screening process that allows for strategic determination of optimally performing primer sets and standardized assay conditions which enable execution of LAMP at point-of-care (POC) settings using complex human samples such as saliva. A total of 20 primer sets targeting the N, E, RdRP, and orf1ab genes of the SARS-CoV-2 were designed, screened, and selected based on performance metrics such as reaction time, sensitivity, and specificity. Of these 20 primer sets, only two primer sets (orf1ab.2 and orf1ab.4) proved to be viable for use in the final assay. Colorimetric RT-LAMP of the selected primer set, orf1ab.2 was shown to produce a distinct color change in contrived samples containing heat-inactivated SARS-CoV-2 in 5% saliva. The limit of detection of our assay using primer set orf1ab.2 was determined to be 1000 copies/µL of saliva collected. Furthermore, methods are introduced which allow for the high-throughput design of LAMP primers using standard software tools and the in-silico performance of LAMP primer sets.