Jun 17, 2026
Version 3
Wastewater Concentration using the Electronegative Filtration method, Total Nucleic Acid Extraction and PCR inhibitor removal for recovery of viral pathogens V.3
- Joyce Akello1,
- Shannon Fitz1,
- Kirsten Williamson2,
- Alex Shaw1,
- Amy Dighe1,
- Billy Thurston1,
- Catherine Pratt2,
- khrisdiana Putri3,
- Daniel Maloney4,
- Omar Khalilur Rahman5,
- Riris Andono Ahmad3,
- Rosanna Glazik6,
- Tony Wawina7,
- Aine OToole4,
- Andrew Rambaut4,
- Jamal Sam5,
- Cheng Siang Tan8,
- Erik Karlsson9,
- Placide Mbala7,
- Indah Kartika Murni3,
- Vicka Oktaria3,
- Sampson Twumasi-Ankrah10,
- Endah Supriyati3,
- michael Owusu10,
- Yoke Fun Chan5,
- Nick Grassly1
- 1Imperial College London;
- 2Biosurv International;
- 3Universitas Gadjah Mada;
- 4University of Edinburgh;
- 5Universiti Malaya;
- 6University of Warwick;
- 7Institut Nationale de Recherche Biomedicale;
- 8Universiti Malaysia Sarawak;
- 9Institut Pasteur Cambodge;
- 10Kwame Nkrumah University of Science and Technology
- Wastewater Surveillance for Pandemic Prevention
Protocol Citation: Joyce Akello, Shannon Fitz, Kirsten Williamson, Alex Shaw, Amy Dighe, Billy Thurston, Catherine Pratt, khrisdiana Putri, Daniel Maloney, Omar Khalilur Rahman, Riris Andono Ahmad, Rosanna Glazik, Tony Wawina, Aine OToole, Andrew Rambaut, Jamal Sam, Cheng Siang Tan, Erik Karlsson, Placide Mbala, Indah Kartika Murni, Vicka Oktaria, Sampson Twumasi-Ankrah, Endah Supriyati, michael Owusu, Yoke Fun Chan, Nick Grassly 2026. Wastewater Concentration using the Electronegative Filtration method, Total Nucleic Acid Extraction and PCR inhibitor removal for recovery of viral pathogens. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzebq8vx1/v3Version created by Joyce Akello
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: June 04, 2026
Last Modified: June 17, 2026
Protocol Integer ID: 318539
Keywords: wastewater, environmental surveillance, WES, WBE, pandemic, RNA viruses, processing wastewater sample, wastewater samples with the goal, magmax wastewater ultra nucleic acid isolation kit, wastewater sample, wastewater ultra nucleic acid isolation kit, electronegative filtration method, nucleic acid extraction, pcr inhibitor removal, wastewater concentration, raw wastewater, total nucleic acid extraction, sewage sample, viral pathogens this standard operating procedure, pcr inhibitor removal for recovery, virus concentration, detecting viral pathogen, zymo onestep pcr inhibitor removal kit, electronegative filtration, extraction, adsorption, principle of adsorption, viral pathogen, ph of the mixture, pcr, standard operating procedure, resulting concentrate, filter
Funders Acknowledgements:
Gates Foundation
Grant ID: PA9917
Institute of Philanthropy
Grant ID: PB3746
Abstract
This standard operating procedure (SOP) sets out the workflow for processing wastewater samples with the goal of recovering and detecting viral pathogens. The steps include virus concentration using the electronegative filtration (ENF) method, total nucleic acid extraction using MagMAX Wastewater Ultra Nucleic Acid Isolation Kit, and PCR Inhibitor removal using the Zymo OneStep PCR Inhibitor Removal Kit.
The raw wastewater/sewage samples are concentrated using the ENF method, which operates off the principle of adsorption-elution. The pH of the mixture is first adjusted, after which the sample is poured through a negatively charged membrane. Viruses are reversibly adsorbed to filters via charge attraction, after which they are eluted into a smaller volume. The resulting concentrate is then subjected to nucleic acid extraction and PCR inhibitor removal.
All procedures can be performed by suitably trained members of staff.
Safety information
Infectious agents – Wastewater contains pathogens, including viruses, bacteria and fungi. Appropriate safety precautions must be exercised when handling wastewater samples.
Materials
Reagents
Equipment
EZ-Fit® Manifold
NAME
Merck
BRAND
EZFITSAM3
SKU
LINK
Hydrochloric acid 1MFisher Scientific UKCatalog #10467640
MagMAX Wasterwater Ultra Nucleic Acid Isolation KitThermo FisherCatalog #A52606
Molecular Biological Grade WaterFisher Scientific UKCatalog #15835408
Ethanol, absolute 99.8%Thermo FisherCatalog #10342652
Zymo OneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030
Equipment
EZ-Stream® vacuum pump
NAME
Merck
BRAND
EZSTREAM1
SKU
LINK
Equipment
EZ-Fit® Manifold Filtration Head
NAME
Merck
BRAND
EZFITMHTA3
SKU
LINK
Equipment
Silicone Tubing
NAME
Merck
BRAND
STREAMTUB
SKU
LINK
Equipment
Fisherbrand™ Microbead Sterilizer
NAME
Fisherbrand
BRAND
16391328
SKU
LINK
Equipment
Bochem™ 18/10 Stainless Steel Forceps, Blunt Tip
NAME
Bochem
BRAND
10169920
SKU
LINK
Equipment
Cole-Parmer BH-250D-4 Dri-Block Digital Block Heater; Quadruple Insert; 100Deg C; 230 VAC
NAME
Scientific Labs
BRAND
BLO1042
SKU
LINK
Equipment
Fisherbrand™ Lab Thermometer
NAME
Fisherbrand
BRAND
11597273
SKU
LINK
Equipment
Thermo Scientific™ HulaMixer™ Sample Mixer
NAME
Thermo Scientific™
BRAND
10548425
SKU
LINK
Equipment
Fisherbrand™ Mini-Centrifuge 100-240V, 50/60Hz Universal Plug, Grey
NAME
Fisherbrand
BRAND
16617645
SKU
LINK
Equipment
DynaMag™-15 Magnet
NAME
DynaMag
BRAND
12301D
SKU
LINK
Equipment
Scientific Industries SI™ Vortex-Genie™ 2
NAME
SI™
BRAND
15557335
SKU
LINK
Equipment
Eppendorf™ Centrifuge 5425/5425 R
NAME
Eppendorf™
BRAND
16845110
SKU
LINK
- Pipettes
- KingFisher Duo System (Optional - if performing automated extractions)
Consumables
- 50mL conical centrifuge tubes, sterile, nuclease-free
- pH indicator strips (pH 0 -14)
- EZ-Fit filtration unit without absorbent pad 0.45 μm pore size (Sigma Aldrich, Catalog #: EFHAW10MS)
- Sterile, RNAse-free pipette tips
- 2.0 ml microcentrifuge tubes
- KingFisher 6-tip comb and 24 DW plate (Thermo Fisher Catalog #97003510. KingFisher‱ Plastics for 24 deep-well format 24 DW tip comb and plate | Buy Online)
- Scienceware vacuum aspirator bottle with trap ( Merck Catalog #: Z675490 ) optional - based on preference for waste disposal)
Preparation and setting up the ENF apparatus.
3m 30s
Turn on the glass bead sterilizer and allow sufficient time for it to reach the operating temperature before use.
Working inside the class II/A2 biological safety cabinet, connect the EZ-Fit manifold to the EZ-Stream vacuum pump (left or right side), then plug the pump into a power source.
3m
Securely mount the EZ-Fit filtration unit onto the EZ-Fit Manifold Base with HTA Manifold Head.
30s
Connect the other side of the vaccum pump to a liquid waste pot/bottle as shown in the figure below. The liquid waste trap or liquid waste pot/bottle can be used and should be connected as shown below ensuring to add virkon/ bleach to the waste trap /pot for disinfection.
Note
The liquid waste trap or liquid waste pot/bottle can be autoclaved and re-used
Sample pH adjustment
3m 30s
Mix the 40 mL wastewater sample thoroughly by inverting several times.
Safety information
Infectious Material - Handle sewage samples in a Class II/A2 biological safety cabinet.
30s
Adjust the sample pH to 3.5 using hydrochloric acid, mixing thoroughly between titrations to ensure a homogeneous solution before proceeding.
3m
Sample Filtration and concentration.
13m 50s
Note
Before adding the 40 mL wastewater sample, verify that the valve is in the horizontal (closed) position
Remove the lid on the EZ-fit filtration unit and carefully pour the 40 mL wastewater sample into the membrane filter.
3m
Place the lid back onto the EZ-fit filtration unit and turn the valve to the vertical position.
10s
Touch the button on top of the EZ-Stream vacuum pump to start filtration
10s
Allow the liquid to flow into the waste container until filtration is complete.
Note
Ensure the entire sample passes through the membrane filter.
30s
Remove the funnel carefully, ensuring the membrane remains on the lower portion of the funnel.
Note
The protective rim minimizes cross-contamination during transfer to a clean 2 mL tube.
30s
Spray two forceps with 70% ethanol and sterilize them by briefly submerging in the glass bead sterilizer.
30s
Using both forceps, gently roll the membrane filter tightly ensuring that the filter is facing outwards and place it into a labelled clean 2 mL tube.
Note
When concentrating multiple samples, after rolling each filter and placing it in a tube, repeat sterilization (step 12) for both forceps prior to moving onto the next sample.
3m
Add 1 mL of lysis buffer from the MagMAX Wastewater Ultra Nucleic Acid Isolation Kit to the tube containing the rolled filter.
2m
Vortex vigorously for 30 seconds to 1 minute to mix thoroughly until the lysate is cloudy and brown indicating that the solids have detached from the filter. Briefly centrifuge to collect the contents.
2m
Transfer the supernatant (lysate) to a clean 2.0 mL tube for immediate extraction. For automated extraction using the KingFisher system, transfer the lysate to the 24-deep-well plate (row A).
2m
Extraction using the MagMAX Wastewater Ultra Nucleic Acid Isolation Kit
2h 1m 50s
Reagent preparation
Prepare 80% ethanol by diluting 100% (absolute) ethanol with nuclease-free water. Prepare a minimum of 4 mL of 80% ethanol per sample.
2m
Bead Mix
Note
Prepare the bead mix on the day of use.
Vortex the Binding Beads vigorously to ensure complete resuspension.
30s
Prepare the Binding Bead Mix by combining the required components below for the total number of samples plus an additional 10% excess.
| Component | Volume per sample | |
| Binding Solution | 2000 μl | |
| Binding Beads | 100 μl | |
| Total volume | 2100 μl |
3m
Mix the Binding Bead Mix well by inversion, then store at room temperature.
10s
Automated extraction on KingFisher Duo
Attached here is the KingFisher Duo Prime protocol for importing into your Instrument.
MagMAX_Wastewater_DUO24.bdz3.5KB
MagMAX_Wastewater_DUO24.bdz3.5KB Note
Minimum number of samples (including controls) for extraction using the 24 DW plate on Duo = 4
Maximum number of samples (including controls) for extraction using the 24 DW plate on Duo = 6
The minimum number of sample is a guide based on the cost of associated consumables
Processing plate set up
Plate 1 containing the samples, tip comb, wash buffer and 80% ethanol
| Row ID | Plate row | Reagent | Volume per well | |
| Sample | A | Sample lysate + Proteinase K + Binding bead mix | 3260 µL | |
| Tip Comb | B | Place a 6-tip comb in the plate | ||
| Wash 1 | C | Wash buffer | 4,000 µL | |
| Wash 2 | D | 80% Ethanol | 4,000µL | |
Plate 2 containing the elution solution
| A | B | C | D | |
| Elution | A | Elution solution | 100 µL | |
| B | Empty | |||
| C | Empty | |||
| D | Empty | |||
Note
To avoid evaporation and contamination, cover the prepared processing plates with
paraffin film or MicroAmp Clear Adhesive Film until they are loaded into the instrument
Transfer 1 mL of lysate to row A of a new 24-deep-well sample plate.
2m
Add 160 µL of Proteinase K to each sample well.
2m
Invert the Binding Bead Mix several times to resuspend the beads, then add 2,100 µL of Binding Bead Mix to each sample in row A.
Note
Keep the Binding Bead Mix well mixed throughout pipetting. Pipette slowly to ensure accurate volumes, and use fresh pipette tips for each addition. Do not reuse tips due to the high viscosity of the mix which will cause variations in the volumes added
5m
Place a 6-tip comb into row B of the 24-deep-well plate. Label this as plate 1
10s
Add 4,000 µL of wash buffer to row C.
4m
Add 4,000 µL of 80% ethanol to row D.
4m
Add 100 µL of elution buffer into a separate 24 deep-well plate in position row A. Label this as plate 2.
1m
Select the MagMAX_Wastewater_DUO24 program on the KingFisher DUO
Start the run, then load the prepared plate 1 and 2 into position when prompted by the instrument
At the end of the run (~28 minutes), immediately remove the plate from the instrument and transfer the eluate to a new 1.5 mL tube.
28m
Keep the isolated nucleic acid on ice to be subjected to PCR inhibitor removal using the Zymo OneStep PCR Inhibitor Removal Kit or store at −80 °C until ready for processing.
Note
See section " Removing of PCR inhibitors using the Zymo OneStep PCR Inhibitor Removal Kit" for the procedure.
Manual Extraction
Note
Minimum number of samples including controls for extraction using manual method = 1
Maximum number of samples including controls for extraction using manual method = 16
Transfer the 1mL lysate to clean 5 mL eppendorf tube
3m
Add 160 µL of Proteinase K to each sample.
2m
Invert the Binding Bead Mix several times to resuspend the beads, then add 2,100 µL of Binding Bead Mix to each sample.
Note
Keep the Binding Bead Mix well mixed throughout pipetting. Pipette slowly to ensure accurate volumes, and use fresh pipette tips for each addition. Do not reuse tips due to the high viscosity of the mix which will cause variations in the volumes added
5m
Transfer the sample tubes on a shaker or sample mixer and set to shake for 5 minutes at 900rpm.
5m
Incubate the sample at 65°C for 5 minutes.
5m
Place the sample tube on a magnetic stand for at least 5 minutes, or until all of the beads have collected.
5m
With the sample on the magnetic stand, carefully remove and discard the supernatant.
Note
IMPORTANT! Avoid disturbing the beads.
5m
Remove the sample from the magnetic stand, then add 4 mL of Wash Buffer to each sample.
2m
Close the tubes and then shake the sample at 800 rpm for 30 seconds on a shaker or sample mixer.
30s
Place the sample tube on the magnetic stand for 3 minutes, or until all of the beads have collected on the side of the tube.
3m
With the tube on the magnetic stand, carefully remove and discard the supernatant
Note
IMPORTANT! Avoid disturbing the beads
3m
Repeat step 19.8 through step 19.11, using 4 mL of 80% ethanol.
8m 30s
Briefly centrifuge and remove any residual solution with a small volume fine-tipped pipette, without disturbing the pellet.
3m
Air-dry the beads by leaving the tube open for 2 minutes to allow residual ethanol to evaporate.
Add 100 μL of Elution Solution to each sample.
2m
Add 100 μL of Elution Solution to each sample and vortex vigorously to fully resuspend the pellet.
3m
Incubate at 75°C for 5 minutes and the shake at 800 rpm for 5 minutes.
10m
Briefly centrifuge and place the sample tube on the magnetic stand for 3 minutes, or until all of the beads have collected to the side of the tube.
3m
With the sample tube on the magnetic stand, carefully transfer the eluates to a new clean 1.5 mL tube.
2m
Keep the isolated nucleic acid on ice to be subjected to PCR inhibitor removal using the Zymo OneStep PCR Inhibitor Removal Kit or store at −80 °C until ready for processing.
Removing of PCR inhibitors using the Zymo OneStep PCR Inhibitor Removal Kit
7m 30s
Preparation of the column
Loosen the screw cap and break the bottom tip of the filter off
30s
Insert column into a Collection Tube.
Centrifuge at 8,000 x g for 3 minutes..
3m
Inhibitor Removal
Transfer the prepared column to a clean 1.5 ml microcentrifuge tube.
1m
Add 100 µl total nucleic acid extract to the Zymo-Spin IV-IR HRC filter and centrifuge at 8,000 x g for 3 minutes.
3m
The filtered total nucleic acid is now suitable for immediate PCR and downstream analysis use or can be store at −80 °C until ready for use.
Protocol references
Solana Narum, Thibault Stalder, Erik Coats, Eva Top 2022. Quantification of the SARS-CoV-2 using electronegative membrane filtration and dPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61xwrvqe/v2 Version created by Solana Narum