Rna minelute clean up

Raw wastewater samples were homogenized and 11 mL were ultracentrifugated as described previously (Wurtzer et al., 2020 (link)). Viral pellets were resuspended in 200 μL of PBS 1×. The membranes of zetapor and nylon were immersed directly in 8 ml of lysis solution to which 4 ml of 1× PBS were added. For virus titration, RNA was extracted from 10 μl of virus stock solution and from 1 ml of exposure mixture. All nucleic acids were extracted using a NucliSENS extraction kit (bioMérieux, Lyon France) as previously described (Vincent-Hubert et al., 2021 (link)), eluted with 100 μl of nuclease-free water and kept frozen at −20 °C until purification. Nucleic acids extracted from membranes exposed in the laboratory were then purified using a Qiagen kit (RNA MinElute Clean up, Qiagen, France) to eliminate potential PCR inhibitors, eluted with 100 μl of nuclease-free water (Qiagen, France) and kept frozen at −20 °C. As partial inhibition of qRT-PCR was found with wastewater samples from field study, we used a One Step PCR Inhibitor removal kit (Zymo Research Kit, USA) for all these samples. Inhibited samples represented 25% and 37% of membranes exposed respectively in seawater and wastewater.