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

LAMP-OSD is designed consciously to be easy to use and interpret, which makes it a reliable choice for either screening or validation of disease states. Base-pairing to the toehold region is extremely sensitive to mismatches, ensuring specificity, and the programmability of both primers and probes makes possible rapid adaptation to new diseases or new disease variants. We have shown that higher order molecular information processing is also possible, such as integration of signals from multiple amplicons 17 . Overall, the use of sequence-specific probes allow construction of strand exchange computation circuits that act as 'matter computers', 5-8 something that is not generally possible within the context of a PCR reaction (which would of necessity melt the computational devices). disruption to standard assay practice. To that end, here we demonstrate the conversion of three recently pre-published LAMP primer sets described for detection of SARS-CoV-2, but that used non-specific readout methods ( Table 1) . The LAMP-OSD versions of these assays maintain the simplicity of visual yes/no readout, while endowing the assays with the inherent accuracy of probe-based signal transduction. We also perform multiplex execution of some of these LAMP-OSD assays and demonstrate the feasibility of sample-to-answer operation by directly analyzing human saliva spiked with SARS-CoV-2 virions.