Quantifying SARS-CoV-2 in Wastewater by RT-qPCR

RT-qPCR was performed directly to quantify the SARS-CoV-2 in the wastewater samples with the premixed primer and probes set 2019-nCoV RUO Kit (Integrated DNA Technologies, Inc., Coralville, IA, USA). A combination of the N1 and N2 primers and probes set was chosen because a study showed that this duplex qPCR assay increased the sensitivity of SARS-CoV-2 detection in wastewater [20 (link)]. The sequences of the primers and probes were found in the study of Qiu et al. [21 (link)]. Each reaction contains the wastewater sample as the template, N1 and N2 primers/probes, and the PowerSYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK). The amplification process was performed with the AB StepOne RT-PCR System (Applied Biosystems, Foster City, CA, USA). Samples were considered positive if amplification was achieved within a cycle threshold of less than 40 cycles (Ct < 40) for at least two out of the triplicates. Dilutions of the 2019nCoV_N Positive Control (Integrated DNA Technologies, Inc., Coralville, IA, USA) were prepared in triplicates to generate a standard curve that was used to calculate the gene copy number (GCN) of SARS-CoV-2.