Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe

Viral evolution impacts diagnostic test performancethrough the emergence of variants with sequences affecting theefficiency of primer binding. Such variants that evade detection bynucleic acid-based tests are subject to selective pressure, enablingthem to spread more efficiently. Here, we report a variant-tolerantdiagnostic test for SARS-CoV-2 using a loop-mediated isothermalnucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating ahigh tolerance to variable SARS-CoV-2 viral sequences, themechanism also overcomes frequently observed limitations ofLAMP assays arising from non-specificamplification withinmultiplexed reactions performed in a single"pot". Results showedexcellent clinical performance (sensitivity 94.5%, specificity 100%,n= 190) when compared directly to a commercial gold standardreverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and thecapability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with nosample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n= 49), openingup the possibility for the assay's use in decentralized testing