Mar 13, 2026
Version 3
Wastewater Concentration Using the Ceres Nanotrap A Particles, Total Nucleic Acid Extraction, and PCR Inhibitor Removal for recovery of viral pathogens V.3
- Shannon Fitz1,
- Joyce Akello1,
- Amy Dighe1,
- Alex Shaw1,
- Billy Thurston1,
- Catherine Pratt2,
- Kirsten Williamson2,
- Khrisdiana Putri3,
- Riris Andono Ahmad3,
- Daniel Maloney4,
- Omar Khalilur Rahman5,
- Rosanna Glazik6,
- Tony Wawina7,
- Aine OToole4,
- Andrew Rambaut4,
- Erik Karlsson8,
- Placide Mbala7,
- Indah Kartika Murni3,
- Vicka Oktaria3,
- Endah Supriyati3,
- Jamal Sam5,
- Cheng Siang Tan9,
- Sampson Ankrah10,
- 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;
- 8Institut Pasteur Cambodge;
- 9Universiti Malaysia Sarawak;
- 10Kwame Nkrumah University of Science and Technology
- Wastewater Surveillance for Pandemic Prevention
Protocol Citation: Shannon Fitz, Joyce Akello, Amy Dighe, Alex Shaw, Billy Thurston, Catherine Pratt, Kirsten Williamson, Khrisdiana Putri, Riris Andono Ahmad, Daniel Maloney, Omar Khalilur Rahman, Rosanna Glazik, Tony Wawina, Aine OToole, Andrew Rambaut, Erik Karlsson, Placide Mbala, Indah Kartika Murni, Vicka Oktaria, Endah Supriyati, Jamal Sam, Cheng Siang Tan, Sampson Ankrah, michael Owusu, Yoke Fun Chan, Nick Grassly 2026. Wastewater Concentration Using the Ceres Nanotrap A Particles, Total Nucleic Acid Extraction, and PCR Inhibitor Removal for recovery of viral pathogens. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq14qxvk5/v3Version created by Shannon Fitz
Manuscript citation:
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: February 09, 2026
Last Modified: March 13, 2026
Protocol Integer ID: 242930
Keywords: wastewater, wastewater surveillance, ceres nanotrap particles, magmax wastewater ultra nucleic acid isolation kit, wastewater ultra nucleic acid isolation kit, wastewater samples for with the goal, processing wastewater sample, total nucleic acid extraction, nucleic acid extraction, wastewater sample, raw wastewater, solution from the extraction kit, ceres nanoparticle, viral pathogens this standard operating procedure, wastewater concentration, detecting viral pathogen, sewage sample, virus concentration, extraction, extraction kit, pcr inhibitor removal, pcr inhibitor removal for recovery, nanotrap buffer, manual extraction, final lysate from the concentration step, viral pathogen
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 for with the goal of recovering and detecting viral pathogens. The steps include virus concentration using the Ceres Nanoparticles A method and total nucleic acid extraction using MagMAX Wastewater Ultra Nucleic Acid Isolation Kit
The raw wastewater/sewage samples are concentrated using the Ceres Nanoparticles A method, adapted to 40 mL starting volume from the Ceres Nanotrap A 35 mL Manual Protocol with MagMAX Wastewater Ultra Nucleic Acid Isolation Kit. The samples are mixed with Nanotrap A particles and Enhancement Reagent 3, incubated on a shaker, and then pelleted on a magnetic rack. The supernatant is removed, and the beads are resuspended in Nanotrap Buffer 2, and pelleted once more. The pellet is then resuspended in lysis binding solution from the extraction kit, incubated at 56°C, and recaptured on the magnetic rack. The final lysate from the concentration step is then removed, leading into automated or manual extraction.
Safety information
Infectious agents – Wastewater contains pathogens, including viruses, bacteria and fungi. Appropriate safety precautions must be exercised when handling wastewater samples.
Guidelines
All procedures can be performed by suitably trained members of staff.
Materials
Nanotrap® Enhancement Reagent 3 (ER3)Ceres NanoCatalog #10113-10
Nanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
Nanotrap® Buffer 2Ceres NanoCatalog #10102-100
MagMAX™ Wastewater Ultra Nucleic Acid Isolation KitThermo Fisher ScientificCatalog #A52606
Equipment
Cole-Parmer BH-250D-4 Dri-Block Digital Block Heater; Quadruple Insert; 100Deg C; 230 VAC
NAME
ScientificLabs
BRAND
BLO1042
SKU
LINK
Equipment
Fisherbrand™ Lab Thermometer
NAME
FisherScientific
BRAND
11597273
SKU
LINK
Equipment
Mini Centrifuge
NAME
FisherScientific
BRAND
16617645
SKU
LINK
Equipment
Thermo Scientific™ HulaMixer™ Sample Mixer
NAME
Thermo Scientific
BRAND
15920D
SKU
LINK
Equipment
DynaMag™-2 Magnet
NAME
Invitrogen
BRAND
12321D
SKU
LINK
Equipment
50 ml Magnetic Separation Rack
NAME
New England Biolabs
BRAND
S1507S
SKU
LINK
Virkon Spray Bottle & Virkon TabletsRMS Industrial and Laboratory Supplies
Ethanol absolute ≥99.8% AnalaR NORMAPUR® ACS Reag. Ph. Eur. analytical reagentVWR International (Avantor)Catalog #20821.330P
UltraPure DNase/RNase-Free Distilled WaterInvitrogen - Thermo FisherCatalog #10977-049
1.5 mL Eppendorf tubes
50 ml Serological Pipette (sterile), individually wrappedStarLabCatalog #E4860-0050 Serological Pipet Controller / PipetboyFisher ScientificCatalog #30245591
OneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6031
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, 6-tip combs and KingFisher 24 deepwell plate (12 pcs of 24 DW plates, each including 4 tips combs)Thermo FisherCatalog #97003510
KingFisher™ Duo and KingFisher™ Duo Prime Consumables, Combi pack for Microtiter 96 Deepwell plate (tips combs, plates and elution strips for 96 samples)Thermo FisherCatalog #97003530
Safety warnings
Experiments MUST be performed in the CL2 within the Class II BSC. Before starting the procedure, ensure that the working area (Class II BSC) and equipment e.g pipettors are thoroughly clean.
Separate dedicated Personal Protective Equipment (PPE) is to be worn in each working area.
Procedure to concentrate viruses
1h 13m
Working inside the class II/A2 biological safety cabinet, mix the 40 mL wastewater sample thoroughly by inverting several times, then let settle (45 seconds at room temperature).
1m
Add 114.3 μL of Nanotrap Enhancement Reagent 3 (ER3) into the sample, close, and invert twice to mix.
1m
Add 600 μL of Nanotrap Microbiome A Particles into the sample, close, and invert twice to mix.
Depicted above: all reagents used in this protocol - Nanotrap A Particles (step 3), Enhancement reagent 3 (step 2), and Nanotrap Buffer 2 (step 7)
1m
Incubate samples combined with Nanotrap A Particles and Enhancement Reagent 3 at room temperature for 30 minutes, inverting every few minutes (can also use a rotator or horizontal shaker).
Can use either rotator (option A) or horizontal shaker (option B). Secure samples in place with tape to avoid falling off if using horizontal shaker. For both rotator or horizontal shaker, secure lids tightly and seal tubes with parafilm prior to commencing 30 minute incubation.
Note
During this incubation, set the heating block to 56°C.
30m
Place samples on the 50 mL magnetic rack for 10 minutes to pellet the Nanotrap particles.
10m
Once 10 minutes are up, remove and discard supernatant with a stripette, taking care to avoid disturbing the pellet. If necessary, use a smaller pipette to remove residual supernatant from the sample.
3m
Resuspend the pelleted Nanotrap particles in 1 mL of Nanotrap Buffer 2, taking care to completely collect all particles.
Transfer the suspension to a clean 2 mL microcentrifuge tube.
5m
Place the sample tubes on a 2 mL magnetic rack to pellet the Nanotrap Particles for 2 minutes.
2m
Carefully remove and discard supernatant using a P-1000 pipette, taking care not to disturb the pellet. If necessary, use a smaller pipette to remove any residual supernatant.
3m
Add 500 μL of MagMAX Wastewater Lysis Solution to the pelleted Nanotrap particles, resuspending thoroughly with the pipette.
2m
Close tubes and incubate for 10 minutes on heating block set to 56°C.
10m
Replace the sample tubes on the 2 mL magnetic rack and allow samples to pellet for 2 minutes.
Note
If necessary, centrifuge samples (2000g for 2-5 seconds) to remove drops from the lid prior to placing on magnet.
2m
For manual extraction, transfer approximately 500 μL of lysate from each sample to a fresh 1.5-2 mL tube and discard pelleted beads.
For automated extraction using the KingFisher Duo, transfer the lysate to the 96-deep-well plate (row A).
Sample is now ready for extraction following either manual or automated small volume extraction workflow.
3m
Extraction using the MagMAX Wastewater Ultra Nucleic Acid Isolation Kit
1h 48m 50s
Reagent preparation
Prepare 80% ethanol by diluting 100% (absolute) ethanol with nuclease-free water. Prepare a minimum of 2 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 | 500 μL | |
| Binding Beads | 20 μL | |
| Total volume | 520 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_DUO96.bdz3.6KB
MagMAX_Wastewater_DUO96.bdz3.6KB Note
Minimum number of samples (including controls) for extraction using the 96 DW plate on Duo = 6
Maximum number of samples (including controls) for extraction using the 96 DW plate on Duo = 12
The minimum number of sample is a guide based on the cost of associated consumables
Processing plate layout
| Row ID | Plate row | Reagent | Volume per well | |
| Sample | A | Sample lysate + Proteinase K + Binding Bead Mix | 1,060 μL | |
| Tip Comb | B | Place a 12 tip comb in the plate | ||
| C | Empty | |||
| Wash 1 | D | Wash Buffer | 1, 000 μL | |
| Wash 2 | E | 80 % Ethanol | 1, 000 μL | |
| Elution | Separate elution strip | Elution Solution | 100 μL | |
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 500 μL of lysate to row A of a new 96-deep-well sample plate.
2m
Add 40 µL of Proteinase K to each sample well.
2m
Invert the Binding Bead Mix several times to resuspend the beads, then add 520 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.
3m
Place a 12-tip comb into row B of the 96-deep-well plate. Leave row C empty.
10s
Add 1,000 µL of wash buffer to row D.
2m
Add 1,000 µL of 80% ethanol to row E.
2m
Add 100 µL of elution buffer into a separate elution strip tube
1m
Select the MagMAX_Wastewater_DUO96 program on the KingFisher DUO
Start the run, then load the prepared plate and elution strip tube 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 tube or plate for downstream analysis or storage.
Note
The isolated nucleic acid is ready for immediate use. For long-term storage, store extracted nucleic acid at −80 °C. Samples may be stored in 1.5 mL tubes.
28m
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 500 µL lysate to labelled clean 1.5 mL tube
3m
Add 40 μL of Proteinase K to each sample tube
2m
Invert the tube of Binding Bead Mix several times to resuspend the beads, then add 520 µL of
Binding Bead Mix to each sample
3m
Transfer the sample tubes on a shaker and set to shake for 5 minutes at 900rpm.
5m
Briefly spin down and place the sample tubes on a magnetic stand for at least 5 minutes, or until all of the beads have collected.
5m
With the sample tube on the magnetic stand, carefully remove and discard the supernatant.
Note
Important! Avoid disturbing the beads.
5m
Remove the tube from the magnetic stand, then add 1 mL of Wash Buffer to each sample
2m
Close the tubes then shake at 800 rpm for 30 seconds.
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 sample tube on the magnetic stand, carefully remove and discard the supernatant.
3m
Repeat step 17.7 through step 17.10, using 1 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.
Note
Avoid overdrying the beads to the point of the pellet cracking as this may lower the efficieincy of nucleic acid recovery.
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.
5m
Shake at 800 rpm for 5 minutes.
5m
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
8m
Preparation of the column
Loosen the screw cap and break the bottom tip of the filter off.
30s
Insert column into a Collection Tube.
30s
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
Based on the protocol APP-082 from Ceres Nanosciences.