2.3. qRT-PCR viral quantification

Samples were tested using a CDC RT-qPCR protocol authorized for emergency use that consists of three unique assays: two targeting regions of the virus’ nucleocapsid gene (N1, N2) and one targeting human RNase P gene (RP) (Catalog # 2019-nCoVEUA-01, Integrated DNA Technologies, IDT, Coraville IA) (^{for Disease Control and Prevention, 2020}). A 5 uL of extracted RNA was added to 15 uL of each assay's reaction mixture containing TaqPath 1-Step RT-qPCR Master Mix, CG (Thermofisher Scientific Waltham MA) and the corresponding primer-probe set (IDT), followed by the recommended thermocycler protocol. Plasmid DNA containing the human RPP30 gene and SARS-CoV-2 in vitro transcribed RNA control (nCoVPC, IDT) were used as positive controls. Water was used as a negative extraction control. Samples were designated positive if all three PCRs were positive above the limit of detection (N1 and N2 for virus, RP for adequate sampling). If the N1 and N2 PCRs were negative, but the RP assay had a Ct value ≥30 or was negative, suggesting inadequate sampling, then the sample was re-extracted. The second result was reported if the RP Ct value was <30 or if both N1 and N2 PCRs were positive regardless of RP Ct value.

The viral load of each sample, in copies/mL, was extrapolated from standard curves generated for each viral assay (N1 and N2) using serial dilutions of nCoVPC (range of 2,000 to 1 × 10⁸ viral RNA copies/mL, Catalog # 10006625, IDT Coraville IA) on every qRT-PCR run performed (^{Table S1}). The average copies/mL between the N1 and N2 assays was used as the final quantitative viral load. We note this viral load reflects copies per mL of extracted RNA. Based on our sample collection and RNA extraction volumes as well as volume of template RNA used in the RT-qPCR (5 uL), this viral load represents the number of viral RNA copies per 5mL of VTM or Shield sample.