May 26, 2026

Extraction and Purification of Total Nucleic Acids from Wastewater

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6jy8zvqe/v1
  • Melissa Pitton1,
  • Rachel E. McLeod1,
  • Lea Carduff1,
  • Ayazhan Dauletova1,
  • Jolinda de Korne-Elenbaas1,
  • Charles Gan1,
  • Camille Hablützel1,
  • Aurélie Holschneider1,
  • Seju Kang1,
  • Guy Loustalot1,
  • Patrick Schmidhalter1,
  • Linda Schneider1,
  • Anna Wettlauffer1,
  • Daniela Yordanova1,
  • Timothy R. Julian1,2,3,
  • Christoph Ort1
  • 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland;
  • 2Swiss Tropical and Public Health Institute, Allschwil, Switzerland;
  • 3University of Basel, Basel, Switzerland.
lara.achermann
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6jy8zvqe/v1
Protocol Citation: Melissa Pitton, Rachel E. McLeod, Lea Carduff, Ayazhan Dauletova, Jolinda de Korne-Elenbaas, Charles Gan, Camille Hablützel, Aurélie Holschneider, Seju Kang, Guy Loustalot, Patrick Schmidhalter, Linda Schneider, Anna Wettlauffer, Daniela Yordanova, Timothy R. Julian, Christoph Ort 2026. Extraction and Purification of Total Nucleic Acids from Wastewater. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jy8zvqe/v1
Manuscript citation:
Pitton, M., McLeod, R.E., Caduff, L. et al. A six-plex digital PCR assay for monitoring respiratory viruses in wastewater. Nat Water 3, 1174–1186 (2025). https://doi.org/10.1038/s44221-025-00503-x
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: March 10, 2026
Last Modified: May 26, 2026
Protocol  Integer ID: 313000
Keywords: purification of total nucleic acid, onestep pcr inhibitor removal kit from zymo research, onestep pcr inhibitor removal kit, pcr inhibitor removal, purification, wastewater this protocol, total nucleic acid, wastewater, extraction, zymo research
Abstract
This protocol describes the extraction and purification of total nucleic acids from wastewater, based on the Wizard® Enviro Total Nucleic Acid Kit from Promega, and PCR inhibitor removal, based on the OneStep PCR Inhibitor Removal Kit from Zymo Research.
Materials
Consumables
  • Wizard® Enviro TNA Kit (Promega: Cat. No. A2991)
  • Promega Eluator Vacuum Elution Device (only connection part used) (Promega: Cat. No. A1071)
  • 95-100% Ethanol (Merck: Cat. No. 1.00983.1011)
  • 2-Propanol (Isopropanol) (Merck: Cat. No. 1.02781.2500)
  • Murine Hepatitis Virus (MHV) viral stock for internal control.
  • 5 mL Eppendorf tube, with snap cap, PCR clean (Huberlab: cat. no. 11.3819.46)
  • 50 mL Conical Centrifuge Tubes, sterile (e.g. Falcon®)
  • Sterile micropipette filter tips – nuclease-free grade (20, 200, and 1000 µL)
  • Serological pipettes 25 mL/50mL (Huberlab: cat. no. 7.760 180/7.768 180)
  • Combitips advanced 1.0 mL (Huberlab: cat. no. 11.4990.23)
  • Combitips advanced 2.5 mL (Huberlab: cat. no. 11.4990.24)
  • Combitips advanced 10 mL (Huberlab: cat. no. 11.4990.26)
  • Zymo One-Step PCR Inhibitor Removal Kit (Zymo Research: Cat No. D6030V)
  • Demi-Spinner Apparatus, in house built constructions (Protocol: Preparation of demi-spinner)
  • Nitrile Gloves
  • Lab coat
  • Permanent marker
  • Meliseptol® rapid (Roth, Cat. No: LK31.3)

Equipment
  • OHAUS Frontier 5000 Serie Micro Centrifuge
  • -80 °C freezer
  • Hettich Rotina 380 Centrifuge (swinging bucket centrifuge)
  • Adolf Kühner Benchtop Lab shaker
  • Integra Pipetboy
  • Vitaris Pipetgirl
  • Pipets 1000µL, 200µL, 20µL, 10µL
  • Liebherr 188cm Laboratory Refrigerator (4°C)
  • Telstar Bio II Advance Plus Safety Cabinet (Class 2)
  • Labnet AccuBlock Digital Dry Bath (heating block, up to 65°C)
  • Vacuum manifold with luer-lock stop-cocks
  • Vacuum pump
  • 3D printed Eluators
  • Corning‱ Polypropylene Centrifuge Tubes 250 mL

Analytical material
  • With this protocol, 45 mL of raw wastewater collected from a wastewater treatment plant (WWTP) influent is processed. For respiratory virus quantification, 24-hour composite samples are collected.
  • To minimize degradation of nucleic acids before extraction, raw wastewater samples should be stored at 4°C after collection.
Safety warnings
Experiments with wastewater and Murine Hepatitis Virus (MHV) pose a potential health risk to the users, and so should always be performed in a laboratory with sufficient safety standards and in accordance with local and institutional policies and guidelines. We suggest at least a Biosafety Level 2 Laboratory, as MHV is a Biosafety Level 2 Pathogen.

MHV should be added within a sterile bench. We recommend always wearing, at a minimum, a lab coat, gloves and safety glasses when working with wastewater and material derived from it to limit risks of exposure.

If equipment is contaminated with wastewater, always spray it with 70% Ethanol or equivalent disinfectant and clean the spill thoroughly. After the work is finished, everything should be cleaned and wiped with 70% Ethanol or equivalent disinfectant.

Solid waste generated through sample processing may be contaminated with MHV-spiked wastewater or material derived from it, and so should be disposed of according to local or institutional guidelines for biohazards. For example, material may require autoclaving prior to disposal. Any liquid waste derived from processing should be collected and disposed of according to local or institutional guidelines. The material contains isopropanol, ethanol, and other constituents in the extraction mixture that may require specific waste disposal instructions. In our laboratory, we have shown that after storing the material for 24 hours, viruses are no longer considered infectious (see Gan et al. 2025 for further information: https://doi.org/10.1016/j.bsheal.2025.09.001).
Scope
This protocol can be followed to extract total nucleic acids from raw wastewater, to be used for quantitative PCR, digital PCR (dPCR) or sequencing applications. Murine Hepatitis Virus (MHV) can be spiked into samples prior to extraction as in-process controls to assess cross-contamination or estimate virus recovery efficiency. Additionally, PCR inhibitor removal is included as a step following extraction. This protocol is optimized for detection of respiratory viruses with dPCR, and performance may differ for other targets or other analytical instruments.
The goal of this procedure is to extract and purify total nucleic acids from 45 mL wastewater and concentrate them to a final extract volume of 80 µL for downstream PCR, dPCR, or sequencing analyses.
Preparation of samples and materials
Prepare the Promega Kit reagents as follows, according to manufacturer's instructions.
  • Column Wash 1: Add 57 mL of 100% 2-Propanol and mix well.
  • Column Wash 2: Add 350 mL of 95%-100% Ethanol and mix well.
Fill two 50 mL tubes with nuclease free water (included in the Promega kit) and pre-heat water to 65 °C using a heating block.
Per wastewater sample, prepare the following consumables:
  • One sterile 50 mL centrifuge tube (i.e., Falcon)
  • One sterile 250 mL bottle (i.e., Corning)
  • One sterile 5 mL tube
Prepare Binding Buffer solutions made from Binding Buffer D and E (included in the Promega Kit reagents) in ratios of both 12:1 (in a 1 L glass bottle), according to Table 1, and 4:1 (in a 50 mL centrifuge tube), according to Table 2, in preparation of the use of both solutions later in the protocol.
Number of samplesBinding Buffer D [mL]Binding Buffer E [mL]
1121
7847
1113211
1518015
1922819
2327623
2732427
3137231
3542035
Table 1: Overview for different sample numbers for 12:1 buffer solution.


Number of samplesBinding Buffer D [mL]Binding Buffer E [mL]
10.40.1
72.80.7
114.41.1
1561.5
197.61.9
239.22.3
2710.82.7
3112.43.1
35143.5
Table 2: Overview for different sample numbers for 4:1 buffer solution.



For each sample, pipet 13 mL of 12:1 buffer solution into clean and labelled 250 mL Corning bottles.
For each sample, pipet 500 µL of 4:1 solution into labelled 5 mL tubes.
For each sample, connect a volume extender to a midi-column, label the midi-column and attach the midi-column to the luer-lock stopcocks on the vacuum manifold.
Sample aliquoting and MHV spiking
If MHV is used for both cross-contamination and recovery efficiency estimations, spike every other sample with MHV. Specifically, starting with the first sample collected from each wastewater treatment plant, every other sample is spiked. From each sample to be spiked, pipet 45 mL wastewater into the corresponding 50mL falcon tube for further processing, using a pipette controller with serological pipet.
Working in the sterile bench, add MHV virus at target concentration of approximately 400’000 genome equivalents, into each sample that should be spiked. The volume of MHV added in µL depends on the concentration of the MHV stock.
Shake the MHV-spiked samples horizontally for 20 minutes at 220 rpm on the benchtop lab shaker.
While the MHV-spiked samples are mixing, aliquot the remaining non-spiked samples.
If you are processing simultaneously a bottle control (BTC), which is a negative control used to assess potential contamination during sample collection, shipping, and processing, pipet 45 mL of the bottle control into a 50 mL tube.
Prepare a full process control (FPC), which is a negative control used to assess potential contamination during sample processing, by filling a 50mL tube with 45mL water directly from the tap, or other source of water without expected contamination of target pathogens.
Lysis and centrifugation
Add 500 µL of Promega Protease solution to each sample, including FPC and BTC, and invert the tube several times to mix.
Incubate the samples for 30 minutes at room temperature. While the samples are incubating, continue preparing materials as needed.
Centrifuge the tubes at 4'843 RCF for 15 minutes in a swing bucket centrifuge to pellet the solid fraction. With a serological pipet, pipet 40 mL of supernatant into the labelled 250 mL Corning bottle without disrupting the pellet.
Caution: If the pellet gets disturbed, repeat the centrifugation step.
Mix the transferred supernatant by shaking the bottle.
Add 48 mL Isopropanol into each 250 mL Corning bottle and mix well by shaking.
Direct capture and purification of total nucleic acids
Make sure all stopcocks of the vacuum manifold are closed and turn on the vacuum pump.

Decant the samples from the 250 mL Corning bottle into the correspondingly labeled column. Invert the bottle on top of the extender to make sure no leftover remains in the bottle.
Open the stopcocks and allow the samples to pass through the filter using the vacuum filtration.
Take the 250 mL bottles off as soon as no more liquid droplets are leaving.
Caution: Be careful that you do not contaminate other samples when removing the 250 mL bottles during this step.
As soon as all of the sample has passed through the column, close the stopcocks again. The stopcocks for samples that have finished filtering, with already dry columns, can be closed earlier to speed up the filtration for the other samples on the same manifold as it increases filtration pressure.
Add 5 mL of Column Wash 1 (CWE) to each column, open the stopcocks, and allow the buffer to flow through. Single columns can be closed as soon as they are empty to speed up the filtration for other columns.
Add 20 mL of Column Wash 2 (RWA) to each column, open the stopcocks, and allow the buffer to flow through. Single columns can be closed as soon as they are empty to speed up the filtration for other columns.
When the buffer has completely passed through all columns, take off the volume extender, open all the stopcocks again and let the columns dry out.
Caution: Make sure there is no buffer residues left in any of the columns, as this can interfere with nucleic acid elution.
Switch off the vacuum pumps but leave the stopcocks open.
Place the labelled 5 mL tubes into the 3D printed eluator device (see Materials, or use the Eluator Device provided by Promega: Eluator‱ Vacuum Elution Device, Cat.# A1071), close the tubes and attach the tubes to the stop-cocks. Place the columns on top. Make sure the outlet of the column is directly on top of the tube.

3D printed eluator with cup in yellow and lid in green.


Add 500 µL nuclease free water that is previously heated up to 65°C directly to the silica membrane. Shake the eluator carefully to make sure the whole membrane is covered with water.
Incubate the silica membrane with water for 1 minute.
Switch on the vacuum pump and let all the water flow through the column. Make sure all the membrane is dry before closing the stop-cock.
When all water has passed through, open all the stop-cocks again and switch off the vacuum pumps.
Repeat steps these elution steps.
Close all the stop-cocks and open them one-by-one to completely dry out the membrane and ensure that no residue water is left. Caution: Always open the stop-cocks slowly and carefully to avoid high pressure within the eluator, as this can lead to splashes in the 5 mL tubes and loss of sample. Additionally, always open the next one first before closing the dry one. If there is always one open, chances of splashes are reduced, as there is never a closed vacuum.
When all columns are dry, close all the stop-cocks and switch off the pumps.
Disconnect and discard the columns.
Carefully remove the 5 mL tubes from the eluators and place them open in a rack.
Add 1.5 mL isopropanol to each sample, close them and mix by inverting the tubes.
The samples can continue to be processed, or be stored at 4°C for up to 24 hours.
Caution: In our experience, storage of samples at 4°C in isopropanol for up to 24 hours does not influence subsequent pathogen detection, but this finding may vary by laboratory.
Purification of total nucleic acids and inhibitor removal
As preparation for the purification, label the following material per sample, and disinfect with UV for 20 minutes:
  • One Demi-Spinner (manual is linked in the material section)
  • 2 mL collection tubes (Promega Kit)
  • 1.5 mL sample tubes with caps for elution (Promega Kit)
  • 1.5 mL sample tubes with caps for zymo column elution (Promega kit or Eppendorf tubes)
Decant the samples into the corresponding Demi-Spinner.
Spin down the 5 mL tubes for 20 sec in the swinging bucket centrifuge to collect residual sample at the bottom (usually ~150 µL).
Pipet the remainder into the appropriate demi-spinner.
Spin the Demi-Spinners at 4'863 x g or 5'000 RPM (max G) in swinging-bucket centrifuge for 6 minutes.
While Demi-Spinners are spinning, precondition the Zymo One-Step PCR Inhibitor Removal spin columns by placing them in a collection tube and adding 600 µL of resin-conditioning “Prep Solution”. Allow this solution to soak the resin in the column for at least 10 minutes at room temperature.
While incubating the Zymo columns, check that all liquid has passed through the Promega PureYield columns inside the Demi-Spinners. If there is any residual liquid, spin those Demi-Spinners for an additional 2 minutes.
Add 300 µL of Column Wash 1 (Promega Kit) and spin the columns at max G for 3 minutes.
Repeat centrifugation if there is any residue.

Add 1'000 µL of Column Wash 2 (Promega Kit) and spin the columns at max G for 4 minutes.
Repeat centrifugation if there is any residue.

Carefully remove the columns from the Demi-Spinner apparatus and snap off the strut. If a column is stuck in the spinner, use a tweezer to take it out. Discard everything except for the column and the cap. The caps of the demi-spinners can be reused after 20 minutes of UV-treatment. Caution: Avoid getting flow-through liquid on gloves to prevent cross-contamination. If gloves get wet, change them to avoid cross-contamination.

Place the columns into labelled 2 mL collection tubes and spin at 10’000 x g for 1 minute to remove residual wash buffer.

Place the columns in labelled 1.5 mL sample tubes.

Add 40 µL nuclease free water (65°C) directly to the silica membrane and incubate for 1 minute.
Centrifuge at 10'000 x g for 1 minute.
Repeat these elution steps to achieve a final elution volume of approximately 80 µL.


Remove and dispose the columns and close the samples.
After the 10 minutes incubation time of the pre-conditioned Zymo columns, spin the columns at 8'000 x g for 3 minutes.
Discard the collection tube and place the Zymo columns into the second set labelled 1.5 mL tubes.
Pipet the eluate into the Zymo columns and spin the columns at 16'000 x g for 3 minutes.
Discard the Zymo columns.
Dilute the samples 3-fold for further dPCR analysis by mixing 20 µL of eluate with 40 µL nuclease free water.
Caution: In our experience, 3-fold dilution of samples increases reliability of downstream digital PCR processing by further decreasing inhibition effects through dilution. Alternative dilutions, including no dilution, can be used based on downstream applications and trade-offs between sensitivity and assay inhibition. For example, wastewater extracts showing elevated inhibition at 3-fold dilution can be instead diluted to achieve a final 5-fold dilution, by adding 80 µL nuclease free water to an aliquot of 20 µL.
The extracts are further analysed with dPCR.
Purification of total nucleic acids and inhibitor removal
Store samples at 4°C for a maximum of 24 hours. For long-term storage we recommend storage at −80°C.
Caution: RNA and DNA decay rates increase at increasing temperatures, however freeze-thaw-cycles of extracts also causes nucleic acid decay. In our experience, short-term storage at 4°C up to 24 hours achieves better results than short-term storage at -80°C, but results may vary by laboratory.
Quality control
Negative controls (Bottle Controls, BTC, and Full Process Controls, FPC) are prepared in this protocol to assess contamination occurring during sample collection, shipment, and processing. MHV spike-in is added to a subset of samples to assess efficacy of the process, acting as a positive extraction control. However, results of the quality control are only obtained after downstream processing, such as quantification of targets with dPCR.
Protocol references
Pitton, M., McLeod, R.E., Caduff, L. et al. A six-plex digital PCR assay for monitoring respiratory viruses in wastewater. Nat Water 3, 1174–1186 (2025). https://doi.org/10.1038/s44221-025-00503-x
Acknowledgements
The work published is based on Pitton et al. (2024) DOI: 10.1038/s44221-025-00503-x
This study was funded by the Swiss National Science Foundation (SNSF Sinergia grant number CRSII5_205933) and by the Swiss Federal Office of Public Health (grant numbers 142006108/334.0-101/26 and 142006655/334.0-107/12) granted to C.O. and T.R.J.

Protocol Prepared for Protocols.io by: Lara Acherman, Eawag, Swiss Federal Institute of Aquatic Science and Technology