Filter flask

Archived wastewater samples (−20 °C) were thawed at 4 °C and viruses were concentrated using the adsorption-extraction (AE) method (Ahmed et al., 2020a ). This method is routinely used in our laboratory for the concentration of microorganisms from environmental water and wastewater samples. The high recovery efficiency of this method for DNA and RNA viruses have been reported previously (Ahmed et al., 2015 (link); Ahmed et al., 2020a ). A 50 mL subsample from each thawed wastewater sample was supplemented with MgCl₂ to achieve a final concentration of 25 mM. After amendment with MgCl₂, 50 mL wastewater samples were immediately filtered through a 0.45-μm pore-size, 47-mm diameter electronegative HA membrane (Cat. No. HAWP04700) (Merck Millipore Ltd., KGaA, Darmstadt, Germany) via a magnetic filter funnel (Pall Corporation, New York, USA) and filter flask (Merck Millipore Ltd.) (Ahmed et al., 2020a ).

Viruses were concentrated from the SARS-CoV-2 seeded wastewater samples using the adsorption extraction (AE) method. This method has been commonly used to concentrate SARS-CoV-2 RNA from wastewater (Ahmed et al., 2020a ; Jafferali et al., 2021 ; Juel et al., 2021 ; Sapula et al., 2021 ). The AE method began with the addition of dissolved MgCl₂ to the sample to achieve a final concentration of 25 mM MgCl₂. After amendment with MgCl₂, wastewater samples were immediately filtered through a 0.45-µm pore-size, 47-mm diameter electronegative HA membrane (HAWP04700; Merck Millipore Ltd, Sydney, Australia) via a magnetic filter funnel (Pall Corporation) and filter flask (Merck Millipore Ltd.) (Ahmed et al., 2020a ). Following filtration, using aseptic technique, the membrane was immediately removed, rolled, and inserted into a 5-mL-bead-beating tube (Qiagen, Valencia, CA) for nucleic acid extraction.

Endogenous viruses were concentrated from the 18 wastewater samples using the AE concentration workflow. The AE workflow began with the addition of dissolved MgCl₂ (Sigma-Aldrich, St. Louis, Missouri, USA) to a 10 mL wastewater sample to achieve a final concentration of 25 mM MgCl₂. After amendment with MgCl₂, wastewater samples were immediately filtered through 0.22, 0.45 and 0.80-μm pore-size, electronegative HA membranes (Merck Millipore Ltd., Tokyo, Japan) via a magnetic filter funnel (Pall Corporation, Port Washington, New York, USA) and filter flask (Merck Millipore Ltd.) (Ahmed et al., 2020b ). Following filtration, using aseptic technique, the membrane was immediately removed, rolled, and inserted into a 5-mL-bead-beating tube (Qiagen, Hilden, Germany) for nucleic acid extraction.