Dec 02, 2021
Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation
- 1FDA
- Jacquelina.Woods: Method Developer
- GenomeTrakrTech. support email: genomeTrakr@fda.hhs.gov
Protocol Citation: Jacquelina.Woods, rachel.rodriguez 2021. Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.bx9ipr4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 15, 2021
Last Modified: December 02, 2021
Protocol Integer ID: 53258
Keywords: GenomeTrakr, wastewater, SARS-CoV-2, virus, concentration, PEG precipitation, ultracentrifugation
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.
Materials
EQUIPMENT AND SUPPLIES:
1. Biological Safety Cabinet (BSC Type A2 or higher air exchange rate)
2. Nalgene PPCO 1L centrifuge bottles (Fisher Scientific, 05-562-25, or equivalent)
3. Adjustable Calibrated Micropipettor (1000 μl)
4. Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
5. DNase/RNase-free microcentrifuge tubes 2.0 mL, non-stick, low retention, siliconized (Life Technologies AM12475)
6. Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 μl)
7. Centrifuge capable of speeds of ≥12,000 x g (e.g., ThermoFisher RC6), and with rotors capable of holding 50ml conical tubes (ThermoFisher Scientific, 36-101-0816, or equivalent)
8. 50ml conical tubes (Fisher Scientific, 14-959-49A).
9. 100mL disposable graduated pipettes (Fisher Scientific, 03-395-177, or equivalent)
10. 50ml disposable graduated pipettes (Fisher Scientific, 13-678-11F, or equivalent)
11. FY14 50 carbon fiber rotor (ThermoFisher Scientific, 46922, or equivalent)
12. Ultracentrifuge capable of speeds of ≥170,000 x g (e.g., Sorvall WX90) and with rotors capable of holding 70ml ultracentrifuge tubes (ThermoFisher Scientific, 75000100, or equivalent)
13. Refrigerated incubator shaker (Eppendorf New Brunswick™ I24R shaker M1344-0014 or equivalent)
14. Refrigerated incubator shaker platform (Eppendorf, M1250-9902, or equivalent)
15. Refrigerated incubator shaker clamps with springs (Eppendorf, ACSB-500S, or equivalent)
16. Carbon Fiber Rotor F40L, 8 x 100 ml:(Thermo Scientific, 096-087057, or equivalent)
17. Carbon Fiber ROTOR SS F10-4X1000Y LEX (Thermo Scientific, 09-604-1053, or equivalent)
18. 70 mL Polycarbonate ultracentrifuge tubes w/aluminum cap tubes for F40L rotor (Fisher Scientific, 010-1333, or equivalent)
19. Vortex mixer (Labsource, S16-200, or equivalent)
20. Mettler Toledo™ NewClassic ME Precision Balance, 2200g (Fisher Scientific, 01-912-408, or equivalent)
21. Disposable transfer pipettes (Fisher Scientific, 13-711-22, or equivalent)
MEDIA AND REAGENTS:
1.GlycineSigma AldrichCatalog #G7126,, or equivalent
2.Guanidine IsothiocyanateThermo FisherCatalog #15535016 , or equivalent
3. Hydrochloric Acid (HCl)Fisher ScientificCatalog #6000710 , or equivalent
4. Nuclease Free WaterLife TechnologiesCatalog #AM9937 , or equivalent
5. Sodium Chloride (NaCl)Sigma AldrichCatalog #S3014 , or equivalent
6. Potassium ChlorideSigma AldrichCatalog #P9541 , or equivalent
7. Potassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791 , or equivalent
8. Sodium Phosphate dibasic anhydrous (Na2HPO4)Sigma AldrichCatalog #S5011 , or equivalent
9. Tris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856,, or equivalent
10. EDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261,, or equivalent
11. 10X Phosphate Buffered SalineSigma AldrichCatalog #P5493 , or equivalent
12. PEG 20000Sigma AldrichCatalog #81275-1KG , or equivalent
Safety warnings
Appropriate BSL-2 controls should be used when handling and processing wastewater samples. All steps for the concentration of viruses from wastewater should be completed in a BSC Type A2 or higher.
Before start
Safety information
If sample is frozen, thaw under refrigeration (2-8 °C ). For an 800 mL sample, thawing may take up to five (5) days.
To preserve RNA viruses, samples should be kept on ice whenever possible during the process. To avoid any RNA degradation, sterile glassware and DNase/RNase-free microcentrifuge tubes should be used.
Virus concentration
Virus concentration
30m
30m
Place 800 mL of wastewater into an 1L centrifuge bottle.
Add 200 µL 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 40 mL of 2X PEG.
Protocol
NAME
2X PEG CREATED BY
Jessica L Jones
Dissolve components in ~700 mL deionized or ultrapure water. Heating to 50 °C is recommended to facilitate dissolution.
200 g PEG-20000Sigma AldrichCatalog #81275-1KG , or equivalent
34.8 g Sodium ChlorideFisher ScientificCatalog #S271,, or equivalent.
Bring total volume up to 1 L with deionized or ultrapure water.
Autoclave 121 °C 00:15:00 .
15m
Store at Room temperature protected from light.
Add 400 µL of 5N HCl.
Protocol
NAME
5N HClCREATED BY
Jessica L Jones
Measure 14.6 mL of deionized or ultrapure water into sterile container.
Slowly add 10.4 mL Hydrochloric Acid (HCl)Fisher ScientificCatalog #6000710 , or equivalent to the water.
Safety information
This solution should be prepared under a chemical fume hood.
Store at Room temperature .
Shake briefly and place bottles in refrigerated shaker at 100 rpm, 6-8°C for 02:00:00 .
2h
Centrifuge 8000 x g, 2-6°C, 00:30:00 .
Note
For ThermoFisher RC6, set acceleration at 7 and deceleration at 3, or equivalent speeds for other models.
30m
Carefully pipette supernatant and discard, care should be taken not to disrupt the pellet.
Note
Up to 15 mL of residual supernatant may remain, it is not necessary to remove this volume and risk disrupting the pellet.
Resuspend pellet in 15 mL of 0.05M glycine and transfer entire volume to a 50 ml conical tube.
Protocol
NAME
0.05 M Glycine
CREATED BY
Jessica L Jones
Mix 3.75 g GlycineSigma AldrichCatalog #G7126 in deionized or ultrapure water to 1 L
Adjust 9.5
Autoclave 121 °C 00:15:00
15m
Store at 2-8 °C
Incubate On ice for 00:40:00 +/- 10 min , vortex occasionally.
40m
Add 15 mL of 2X tc PBS to neutralize. Shake by hand to mix.
Protocol
NAME
Tissue Culture (tc) PBS
CREATED BY
Jessica L Jones
Dissolve components in deionized or ultrapure water to 1L.
8.0 g Sodium Chloride (NaCl)Sigma AldrichCatalog #S3014
0.2 g Potassium ChlorideSigma AldrichCatalog #P9541
0.12 g Potassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791
0.91 g Sodium Phosphate dibasic anhydrous (Na2HPO4)Sigma AldrichCatalog #S5011
Adjust 7.5
Autoclave 121 °C 00:15:00
15m
Store at 2-8 °C
Note
For 2X tc PBS, double components in 1.1 - 1.4
Note
Alternatively, 1X and 2X PBS can be made by dilution of 10X PBSSigma AldrichCatalog #P5493 into sterile deionized or ultrapure water.
Centrifuge at 3000 x g, 2-6°C, 00:15:00 .
15m
Transfer supernatant to clean ultracentrifuge tube (careful not to disturb pellet).
Bring total volume up to 65 mL or 125 g total weight (includes bottle and cap) with addition of 1X tc PBS.
Protocol
NAME
Tissue Culture (tc) PBS
CREATED BY
Jessica L Jones
Dissolve components in deionized or ultrapure water to 1L.
8.0 g Sodium Chloride (NaCl)Sigma AldrichCatalog #S3014
0.2 g Potassium ChlorideSigma AldrichCatalog #P9541
0.12 g Potassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791
0.91 g Sodium Phosphate dibasic anhydrous (Na2HPO4)Sigma AldrichCatalog #S5011
Adjust 7.5
Autoclave 121 °C 00:15:00
15m
Store at 2-8 °C
Note
For 2X tc PBS, double components in 1.1 - 1.4
Note
Alternatively, 1X and 2X PBS can be made by dilution of 10X PBSSigma AldrichCatalog #P5493 into sterile deionized or ultrapure water.
Balance tubes to within 0.05 g of each other using 1X tc PBS.
Centrifuge at 170000 x g, 2-6°C, 00:45:00 .
Note
Minimum volume for ultracentrifugation using Fiberlite rotor and tubes is 50 mL.
45m
Discard supernatant and resuspend pellet in 800 µL of 1X tc PBS.
Evenly distribute sample into four 2.0 ml microcentrifuge tubes.
Store concentrates from Step 17 at -70 °C or proceed directly to RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits (protocols.io).
Note
Only 1 tube is required for RNA extraction. The remaining tubes should be stored at -70 °C as reserve.