2.3. Viral RNA extraction and RT-qPCR

RNA extraction for the 107 sewage samples was conducted using the Trizol plus RNA Purification Kit (Thermofisher) following the manufactures' protocols. After RNA extraction, a one-step N-gene-specific RT-qPCR (^{Chu et al., 2020; Pan et al., 2020; Vogels et al., 2020}), was then performed for the detection of viral signals (Table S1). The one-step RT-qPCR was carried out for 45 cycles with a 20 μL reaction mixture using the TaqMan Fast Virus one-step Master Mix (Thermo Fisher, USA). In a typical 20 μL reaction, it contains 5 μL of 4 × TaqMan Fast Virus 1-Step Master Mix, 500 nM of forward primer, 500 nM of reverse primer, 250 nM of probe, and 4 μL of RNA template were used. The RT-qPCR condition was as follows: 50 °C for 5 min, 95 °C for 20 s, 45 cycles at 95 °C for 5 s and 58 °C for 30 s. If the cycle threshold (Ct) value of a sewage sample was ≤45, the sample was considered to have a SARS-CoV-2 signal. To quantify the copy number of the viral RNA, a standard curve was generated by using the serially diluted plasmid DNA containing the target gene (10 to 10⁷ copy/reaction). The quantification limit of the assay was 10 copy/reaction. The primer-probe set used in the N-gene-specific RT-qPCR for the detection of the SARS-CoV-2 RNA enabled the detection of SARS-CoV-2 viral RNA. The standard curve has a dynamic range of 10–10⁷ copy/reaction, and good correlation coefficient (R²) (0.99–1). For quality assurance and quality control (QA/QC), the reagents in the RNA extraction kit were used as the negative control (as the “reagent blank”) for RNA extraction and quantification steps. The no-template control (NTC) was included as a negative control for RT-qPCR. For one of the samples, the RT-qPCR product was sent out for Sanger sequencing at the Beijing Genomics Institute (BGI) to get the sequence of the product.