Mar 20, 2026

Public workspaceConcentration and extraction of wastewater samples - 35mL

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5knk6v1b/v1
  • Catherine Pratt1,
  • Kirsten Williamson1
  • 1Biosurv International
Kirsten Williamson
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5knk6v1b/v1
Protocol Citation: Catherine Pratt, Kirsten Williamson 2026. Concentration and extraction of wastewater samples - 35mL. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5knk6v1b/v1
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 08, 2025
Last Modified: March 20, 2026
Protocol Integer ID: 226753
Keywords: mpox, WES, wastewater, surveillance, ceres, extraction, environmental, grab, samples, concentration of mpox virus particle, mpox virus particle, extraction of wastewater sample, ceres nanosciences nanotrap microbiome bead, wastewater sample, thermofisher magmax wastewater kit, wastewater environmental sample, detection of viral pathogen, nucleic acid extraction, nucleic acid suitable for downstream qpcr, viral pathogen, rich wastewater matrice, nucleic acid
Funders Acknowledgements:
Gates Foundation
Grant ID: INV-095767
Gates Foundation
Grant ID: INV-076176
Abstract
This protocol describes the concentration of Mpox virus particles from wastewater environmental samples using the Ceres Nanosciences Nanotrap Microbiome beads, followed by nucleic acid extraction using the ThermoFisher MagMAX Wastewater kit. It is optimized for environmental surveillance applications, including the detection of viral pathogens in low-copy, inhibitor-rich wastewater matrices.

The procedure yields purified nucleic acid suitable for downstream qPCR or sequencing, with improved sensitivity compared to direct extraction approaches, and the inclusion of an internal control enables monitoring of remaining inhibition.
Materials
Reagents
NameSupplierCatalog Number
Nanotrap Enhancement Reagent 1 (ER1)Ceres Nanosciences10111
Nanotrap Microbiome A ParticlesCeres Nanosciences44202
Nanotrap Buffer 2Ceres Nanosciences10102
Nuclease-free water (not DEPC-treated)ThermoFisher ScientificAM9937 or alternate
MagMAX Wastewater Ultra Nucleic Acid Isolation KitThermoFisher ScientificA52606
100% Ethanol (non-denatured)variousvarious
Internal Controlvariousvarious

Equipment & Consumables

Equipment:
  • Heat block for 1.5mL tubes
  • Vortex
  • Microcentrifuge (1.5mL tubes, strip tubes)
  • Pipettes (p20, p200, p1000)
  • Pipette boy
  • Tube rack for 50mL tubes
  • Tube rack for 1.5ml tubes
  • Magnetic rack for 50mL tubes
  • Magnetic rack for 1.5mL tubes

Consumables:
  • 50mL falcon tubes
  • 1.5mL DNA LoBind tubes
  • Vortex
  • Pipette filter tips (20uL, 200uL, 1000uL)
  • Serological pipettes (25mL)

Safety:
  • Biosafety cabinet
  • Disposal bag/bin for pipette tips
  • Disposal bag/bin for 25ml serological pipettes
  • Disposal method for liquid waste containing infectious material
  • Disposal method for liquid waste containing guanidine thiocyanate

For automated extraction with KingFisher Duo Prime only (available from ThermoFisher Scientific):
Equipment:
  • KingFisher Duo Prime with 12-pin magnetic head and heat block (5400110)

Consumables:
  • KingFisher Combi Pack (97003530)
or
  • KingFisher 96 deep well Plate, v-bottom (95040450)
  • KingFisher 12-Tip Comb (97003500)
  • KingFisher Elution Strip (97003520)
  • KingFisher Elution Cap (97003540)

For automated extraction with KingFisher Flex only (available from ThermoFisher Scientific):
Equipment:
  • KingFisher Flex (5400630)
  • KingFisher Flex 96 deep well magnetic head (24074430)
  • KingFisher Flex 96 deep well heat block (24075430)

Consumables:
  • KingFisher 96 deep well plate, v-bottom (95040450)
  • KingFisher 96 deep well tip comb (97002534/97002820)
Troubleshooting
Safety warnings
Handling potentially infectious material Wastewater samples may contain infectious pathogens. All sample handling should be conducted using appropriate biosafety procedures and personal protective equipment (PPE). Work should be performed in accordance with institutional biosafety guidelines and, where possible, within a biological safety cabinet to reduce aerosol exposure. Avoid generating aerosols and disinfect work surfaces before and after processing.

Guanidine Thiocyanate (Lysis buffer, Binding Solution, Wash buffer) Guanidine thiocyanate is a strong chaotropic agent that is harmful if inhaled, ingested, or absorbed through the skin. Always handle with appropriate PPE. Never mix guanidine-containing reagents with bleach (sodium hypochlorite) as this can produce highly toxic gases. Dispose of guanidine-containing waste according to institutional chemical waste procedures.

Ethanol Ethanol is highly flammable. Keep away from open flames, sparks, and hot surfaces. Use in well-ventilated areas and keep containers tightly closed when not in use. Store in appropriate flammable-liquid cabinets where required by local safety regulations.

Bleach (Sodium Hypochlorite) Bleach is corrosive and can cause skin and eye irritation. Wear appropriate PPE when preparing or using bleach solutions. Use in a well-ventilated area. Do not mix bleach with guanidine-containing reagents, acids, or ammonia, as this may generate toxic gases.

Sample Preparation - work in a biosafety cabinet
1h
Pre-heat a 1.5mL tube heat block to Temperature56 °C
Label 1 x 50mL tube and 1 x 1.5mL tube for each sample
Follow the sub-step below to spike the internal control into the lysis buffer. If you are not using internal control, skip this step.
Prepare the mastermix below for the number of samples and controls plus 10%:
ReagentVolume (uL)
MagMAX Wastewater Lysis Buffer500
Internal Control (10,000 copies/uL)2.5

Invert wastewater samples 5 times to mix. Let sit for 45 second at TemperatureRoom temperature
45s
Using a serological pipette, aliquot 35mL of each wastewater sample into the labelled 50mL falcon tube
Viral capture using Ceres beads - work in a biosafety cabinet
59m
Add 100μL of Nanotrap Enhancement Reagent 1 (ER1) to each sample

Close the lids securely and invert 5 times to mix
Add 525μL of Nanotrap Microbiome A particles to each sample
Close the lids securely and invert 3-5 times to mix
Incubate at TemperatureRoom temperature forDuration00:30:00 .
Note
Invert 3 times to mix, every 5 minutes, during the incubation


30m
Place the tube on a 50mL magnetic rack for Duration00:10:00 or until beads have separated from the solution

10m
Using a serological pipette, discard the supernatant carefully, without disturbing the pellet
Use a 1mL pipette to remove any remaining liquid from each of the 50mL tubes
Add 1mL of Nanotrap Buffer 2 OR nuclease-free water to each pellet

Note
Wet the beads as you add the buffer/water. Add to all samples before moving onto mixing to avoid over-drying of beads.

Pipette to mix, or close the lids securely and swirl the tubes, to resuspend the beads.

If beads remain stuck to the tube wall, use a micropipette to aspirate liquid from the bottom of the tube and then gently dispense the liquid against the tube wall to wash the beads off the wall.
Using a 1mL pipette, transfer the bead suspension into the labelled 1.5mL tube. Be sure to transfer the whole volume, so no liquid remains in the 50mL tube.
Place the tubes on a 1.5mL magnetic rack for Duration00:02:00 or until the beads have separated from the solution

2m
Using a 1mL pipette, discard the supernatant carefully without disturbing the pellet.
Use a smaller pipette to remove the last of the supernatant from the tube, without disturbing the pellet.
Remove the tubes from the magnet and add 500μL of MagMAX Lysis Buffer (prepared with the internal control, if used) from the MagMAX Wastewater Ultra Nucleic Acid Isolation Kit and pipette up and down until fully resuspended

Close the lids securely and verify that each lid is closed. Incubate the samples on the heat block at Temperature56 °C for Duration00:10:00

10m
Centrifuge the tubes for 3 seconds
Place the tubes on a 1.5mL magnetic rack for Duration00:02:00 or until beads have separated from the solution

2m
Proceed to Extraction immediately, where possible.

If not possible, transfer 400uL of the Ceres-concentrated sample into the appropriate plastic consumable for the nucleic acid extraction step:
- 1.5mL tube for manual extraction
- 96 Deep Well plate for automated extraction on a Kingfisher instrument.

Discard the 1.5mL tube containing the Nanotrap particle pellet.

Note
SAFE STOPPING POINT
After concentration, samples mixed with lysis buffer may be stored at Temperature4 °C for up to Duration24:00:00 prior to extraction. Extraction on the same day is strongly recommended to maximize nucleic acid recovery.



Note
Extraction can be performed either manually or using an automated KingFisher instrument. Here we include the protocols for:
  • Manual extraction
  • KingFisher Duo Prime (up to 12 samples per run)
  • KingFisher Flex (up to 96 samples per run)

Step case

Automated Extraction on the KingFisher Duo Prime
13 steps

Use this protocol for automated extraction of up to 12 samples per run on the KingFisher Duo (including controls)
Preparation for extraction - work in a clean area/mastermix room
45m
Before beginning:

i. Check that the KingFisher Duo has the 12-pin magnet and 12 position heat block installed
ii. Check that the KingFisher Duo has the 'MagMAX_Wastewater_DUO96.bdz' protocol script installed
Prepare 1.5mL of 80% Ethanol in nuclease-free water for each sample and control. For example:

If extracting 11 samples + 1 extraction control = 12 x 1.5mL = 18mL of 80% Ethanol required
18mL x 0.8 = 14.4mL of 100% Ethanol
18mL x 0.2 = 3.6mL of nuclease-free water
Prepare the Binding Bead Mix for the number of samples and controls plus 10%. Mix the Binding Bead Mix well by slowly inverting 5-10 times. Do not pipette to mix. For example:

If extracting 11 samples + 1 control = 12 plus 10% overage = prepare Binding Bead Mix for 14 samples

Vortex the Binding Beads well before use. Pipette the reagents very slowly as they are viscous. Do not pipette to mix or to rinse the tip after addition.
ReagentVolume per sample (uL)
MagMAX Binding Beads20
MagMAX Binding Buffer500
Total520

Add the following reagents to each required well of a row in the 96 Deep Well plate. Each sample requires one well in each row, for example 10 samples would take 10 wells in the row. Seal the plate after addition with parafilm or an adhesive plate seal to reduce evaporation.
RowReagents
A-
B12 - Tip Comb
C-
D1mL MagMAX Wash Buffer
E1mL freshly prepared 80% Ethanol
F-
G-
H-

Add 100μL of MagMAX Elution Buffer to each tube in the Elution Strip. Close the strip tubes to reduce evaporation. Remember to remove the lids before loading into the KingFisher Duo.
Extraction - work in a biosafety cabinet
1h 30m
Transfer the prepared plate from the clean area to the biosafety cabinet.
Add 400μL of the concentrated wastewater/lysis supernatant from the Ceres concentration step to row A, using a separate well for each sample.
Add 400μL of nuclease-free water to an empty well in row A to act as the extraction control.
Mix the Binding Bead Mix well by inverting 5-10 times, then add 520μL of the Binding Bead Mix to each sample including the extraction control. Do not pipette to mix with the sample. The KingFisher Duo will perform the mixing.

Do not pipette to mix the Binding Bead Mix, only invert. Use a fresh tip per sample when adding to the plate as the mix is extremely viscous and tip re-use may lead to varied volume addition. Invert to mix if the beads begin to settle during addition.

Add 40μL of Proteinase K to each sample including the extraction control. Do not pipette to mix with the sample. The KingFisher Duo will perform the mixing.
Run the 'MagMAX_Wastewater_DUO96' script on the KingFisher Duo and load the plate and elution strip when instructed
At the end of the run, immediately remove the plate. Seal and centrifuge the plate if possible.
Aliquot extracted nucleic acid into 2 x 50μL aliquots:
One aliquot for PCR analysis, which can be used immediately or stored at Temperature4 °C DurationOvernight
One aliquot for Sequencing, which should be stored at Temperature-20 °C

Note
Although the nucleic acid is eluted into 100uL, there may not be sufficient volume to prepare 2x50uL aliquots due to evaporation during the elution step. In that case, aliquot 1x50uL for PCR, and store the remaining volume for sequencing.

Protocol references
The concentration protocol is adapted from APP-082 from Ceres Nanosciences.
The extraction protocol is adapted from MAN0025693 from ThermoFisher Scientific.