Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation
Jacquelina.Woods, rachel.rodriguez
Abstract
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.
Before start
Steps
Virus concentration
Place 800mLof wastewater into an 1L centrifuge bottle.
Add 200µL of extraction control (concentration of 102 per mL), as described here: Preparation of Murine Norovirus for Use as an Extraction Control for Concentration of Viruses from Wastewater (protocols.io).
Add 40mL of 2X PEG.
Add 400µL of 5N HCl.
Shake briefly and place bottles in refrigerated shaker at 100rpm for 2h 0m 0s.
Centrifuge 8000x g,2-6°C.
Carefully pipette supernatant and discard, care should be taken not to disrupt the pellet.
Resuspend pellet in 15mL of 0.05M glycine and transfer entire volume to a 50 ml conical tube.
Incubate On icefor 0h 40m 0s, vortex occasionally.
Add 15mL of 2X tc PBS to neutralize. Shake by hand to mix.
Centrifuge at 3000x g,2-6°C.
Transfer supernatant to clean ultracentrifuge tube (careful not to disturb pellet).
Bring total volume up to 65mL or 125gtotal weight (includes bottle and cap) with addition of 1X tc PBS.
Balance tubes to within 0.05g of each other using 1X tc PBS.
Centrifuge at 170000x g,2-6°C.
Discard supernatant and resuspend pellet in 800µL of 1X tc PBS.
Evenly distribute sample into four 2.0 ml microcentrifuge tubes.
Store concentrates from Step 17 at -70°Cor proceed directly to RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits (protocols.io).
