2.5. SARS-CoV-2 RNA extraction and viral quantification

RNA extraction of the concentrated viral filtrate was conducted using an AllPrep PowerViral DNA/RNA Kit (Cat. No. 28000-50; QIAGEN, Germantown, MD, USA) following the manufacturer's protocol. 10 μL of total RNA was then reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat No. 4368814). The resulting cDNA was then used for SARS-CoV-2 gene quantification with ddPCR assays.

By targeting the nucleocapsid (N) gene and the envelope (E) gene of SARS-CoV-2 (Table 2 ), the N gene assays employed two primer and probe sets, one from the China Centers for Disease Control and Prevention (CN-CDC) (Suo et al., 2020) and one from the United States CDC (US-CDC) (Hirotsu et al., 2020; Jung et al., 2020; Wu et al., 2020). The ddPCR assay used for the E gene is based on the E_Sarbeco primers and probe set (Corman et al., 2020) recommended by the World Health Organization (WHO). Gene amplifications were conducted using 20 μL reactions containing ddPCR supermix for probes (Bio-Rad, cat No. 1863024), DNase- & RNase-free water, 900 nM of forward and reverse primers, 250 nM of probe, and cDNA templates.

Primers and probes used in the SARS-CoV-2 ddPCR assays.

Parallel to the gene quantifications of the MST targets, droplet generation using the QX200 Droplet Generator (Bio-Rad) was followed by amplification of SARS-CoV-2 genes using a Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following conditions: 94 °C for 10 min, 40 cycles of denaturation and annealing/extension at 94 °C for 30 s and 60 °C for 60 s, respectively, followed by 98 °C for 10 min and then a final hold of 4 °C. Following amplification, target gene concentrations were determined using a QX200 droplet reader (Bio-Rad) and QuantaSoft (V 1.7; Bio-Rad). The limit of detection (LOD) for all assays conducted in this study was 667 GC/L. For a sample to be considered SARS-CoV-2 positive, a single gene, either E or one of the two detection pathways for the N2 gene, must be detected in the samples with concentrations greater than the LOD.