2.3. Viral RNA extraction and qPCR analysis

Viral RNA was purified from the concentrated samples using ABIOpure Viral DNA/RNA Extraction kits (Alliance Bio Inc., Cat#M561VT50) according to the manufacturer instructions. Internal extraction control RNA provided with the RT-qPCR kit was added to the samples before the RNA extraction in order to verify the successful extraction in case of a negative SARS-CoV-2 result. RNA was eluted in a final volume of 40 μL of elution buffer. The viral load in the samples was determined by RT-qPCR using GENESIG COVID-19 kits (Primerdesign Ltd., Cat#Z-Path-2019-nCoV) according to the manufacturer instructions. The kit uses proprietary primers and probes that are directed against the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) gene. The qPCR kit specification sheet states that the detection limit of the kit is 0.58 copies/μL, where 8 μL of input is used in a 20 μL reaction.

An Alternative RNA extraction protocol using TRIzol was also performed. Briefly, 300 μL of chloroform was added to the 1.5 mL of TRIzol re-suspended pellet. The mixture was vortexed for 1 min and incubated at room temperature for 3 min. Samples were then centrifuged at 12,000 ×g for 10 min at 4 °C, and the top aqueous layer was transferred to a clean tube. Isopropanol (1 mL) was added to the aqueous layer and the mixture was agitated by inverting the tube before incubation at room temperature for 10 min. The RNA was pelleted by centrifugation at 12,000 ×g for 15 min at 4 °C and washed once in 75% ethanol prior to resuspension in elution buffer (40 μL) from the ABIOpure Viral DNA/RNA Extraction kit. The two protocols described for viral concentration and RNA extraction (either ultrafiltration columns/RNA extraction kit or PEG/TRIzol) were selected and tested because they were the two methods that were reported for this type of work at the time these experiments were initiated in early April 2020 (^{Wu et al., 2020b; Medema et al., 2020a}). The authors wanted to compare them to decide which methodology to adopt as wastewater sampling continued. All the data reported in the tables were obtained using the ultrafiltration method (Fig. 1a).

Schematic representation of the sample collection, virus deactivation, and RNA concentration and extraction (WWTPs: Wastewater treatment plants, WW: Wastewater) (a) viral concentration using ultrafiltration columns and (b) Viral concentration using PEG-mediated precipitation.

The extracted RNA (8 μL) was used as an input in 20 μL qPCR reactions in 96-well plates on the Viia™ 7 Real-Time instrument by Applied Biosystems. Elution buffer (8 μL) was used a negative control. Standard curves were established using positive control standards provided in the kit. These standard curves were used to determine quantities (in gene copies/μL) from the cycle threshold (C_T) values determined for each sample. Internal RNA extraction controls provided with the qPCR kit verified that water samples that tested negative for SARS-CoV-2 were not false negatives due to the RNA extraction procedure. All samples were tested in triplicate and the average quantities were reported in viral gene copies/L ± standard deviation. The schematic presentation of the complete process (i.e., sample filtration, concentration, and RNA extraction) is illustrated in Fig. 1. The viral load (viral gene copies/L of wastewater) was determined (Eq. (1)):