Standard curve, LOD, and LOD detection rates

Standard curve was generated by spiking a known amount of heat-inactivated virus into healthy donor saliva and then serially diluting this starting sample with healthy human saliva, as indicated for the respective assay. LOD was calculated using the mean and SD of the zero control (blank) and lowest concentration standard following the formula: LOD = mean_blank + 1.645_(SDblank) + 1.645_{(SD low concentration sample)} (24). To assess detection rates near the estimated LOD, healthy donor saliva was spiked with heat-inactivated SARS-CoV-2 virus at 0.25 copy/μl and serially diluted to 0.1 and 0.05 copy/μl concentrations. RNA was extracted from each of these dilution samples by three different individuals to generate three RNA batches. For RT-RPA CRISPR-FDS assays, each extracted RNA batch was analyzed in 20 replicates for a total of 60 replicates among the three batches generated at each concentration. A positive sample was defined as any sample with a CRISPR-FDS signal that was greater than the mean signal of the negative control plus three times its SD.