The eDNA was sampled using two different methods. The first method used a 47‐mm‐diameter glass microfiber filter GF/F (nominal pore size 0.7 μm; GE Healthcare Life Science, Little Chalfont, UK) to estimate eDNA shedding and decay rates, and the second method used a series of 47‐mm‐diameter polycarbonate membrane filters (pore size 10, 3, 0.8, and 0.4 or 0.2 μm; MILLIPORE, US) to estimate eDNA size distribution. Disposable gloves were worn when collecting water samples, and the outside of the sampling bottles was washed with tap water after the samples were collected. This was to prevent contamination during water sampling and filtration. The filtering devices (i.e., filter funnels [Magnetic Filter Funnel, 500 ml capacity; Pall Corporation, Westborough, MA, USA], plastic holders [ADVANTEC, Japan], nipple joints [ADVANTEC, Japan], hoses [TOYOX, Japan], 1‐L beakers, tweezers, and sampling bottles used for water sampling) were bleached after every use in 0.1% sodium hypochlorite solution for at least 5 min.
Magnetic filter funnel
Manufactured by Pall Corporation
Sourced in United States, Japan, Germany, Australia
The Magnetic Filter Funnel is a laboratory equipment designed for efficient filtration processes. Its core function is to provide a controlled and consistent method for separating solids from liquids using magnetic force. The funnel features a built-in magnetic separator that allows for the rapid and precise removal of magnetic particles during the filtration process.
Automatically generated - may contain errors
4 protocols using magnetic filter funnel
1
Estimating eDNA Shedding and Size Distribution
2
Concentrating Endogenous Viruses from Wastewater
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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.
3
Wastewater Virus Concentration via AE Method
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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 ).
4
Wastewater SARS-CoV-2 Concentration by Adsorption
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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.
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