Swab collection solutions optimized for direct addition.

(A) RT-qPCR of N gene RNA or human cell RNA in swab collection solutions. RNA was diluted to the indicated concentration in Solution 1 (10 mM Tris, pH 8, 1 mM EDTA, 0.2% Tween 20, 0.2 mM DTT), Solution 2 (10 mM Tris, pH 8, 1 mM EDTA, 0.5% Tween 20, 0.5 mM DTT), or water, and 10 μL of each dilution were analyzed in 20 μL TaqPath reactions containing the indicated probes. (B) Direct addition to RT-qPCR of cultured SARS-CoV-2 heat-inactivated with or without proteinase K treatment in either water or Solution 2 (10 mM Tris, pH 8, 1 mM EDTA, 0.5% Tween 20, 0.5 mM DTT). 13.5 μL of each sample was added to a 20 μL TaqPath reaction. (C) Comparison of viral detection by direct addition or RNA extraction with the MagMax Viral RNA isolation kit (Thermo Fisher). Cultured SARS-CoV-2 was diluted to the indicated number of infectious units into 0.4 mg/mL proteinase K in water. RNA was analyzed using TaqPath master mix and the N1 primer/probe mixture, either by direct addition of 13.5 μL of heat-inactivated sample to a 20 μL reaction or by addition of 5 μL of purified RNA to a 20 μL reaction. (D) Stability of viral RNA in contrived swab samples in PK collection solution. Cq values from TaqPath RT-qPCRs with the N1 probe for virus alone in 1x DNA/RNA Shield (black points) or virus mixed with human nasal fluid, diluted into proteinase K solution, and allowed to incubate for different amounts of time at room temperature prior to heat-inactivation (red points) or inactivation with an equal volume of 2x DNA/RNA Shield (blue points). Results for two different concentrations of virus are shown.