Mar 16, 2026

Quantification of the Inhibition in the Six-plex Digital PCR Assay targeting SARS-CoV-2 (N1 and N2), MHV, IAV, IBV, and RSV

This  protocol  is a draft, published without a DOI.
  • Melissa Pitton1,
  • Rachel E. McLeod1,
  • Lea Caduff1,
  • 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.
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Protocol Citation: Melissa Pitton, Rachel E. McLeod, Lea Caduff, 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. Quantification of the Inhibition in the Six-plex Digital PCR Assay targeting SARS-CoV-2 (N1 and N2), MHV, IAV, IBV, and RSV. protocols.io https://dx.doi.org/
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: February 17, 2026
Last Modified: March 25, 2026
Protocol  Integer ID: 243378
Keywords: PCR inhibition, RESP6 assay, SARS-CoV-2, dPCR, Wastewater monitoring, determining pcr inhibition, pcr inhibition in the resp6, pcr inhibition, plex digital pcr assay, digital pcr assay, targeting sar, quantification of the inhibition, inhibition calculation, unspiked mastermix on the resp6, dpcr mastermix
Funders Acknowledgements:
Swiss National Science Foundation
Grant ID: CRSII5_205933
Swiss Federal Office of Public Health
Grant ID: 142006108/334.0-101/26
Abstract
This protocol describes the procedure for determining PCR inhibition in the RESP6 digital PCR assay, using SARS-N1 as a representative target. The assay was developed within the context of Pitton et al. (2024) DOI: 10.1038/s44221-025-00503-x. To assess inhibition, a known quantity of synthetic SARS-N1 RNA is spiked into the dPCR mastermix. The inhibition calculation entails a two-run approach: first, SARS-N1 is quantified using an unspiked mastermix on the RESP6, followed by a second run with a spiked mastermix as described in this protocol. The percentage of signal lost because of PCR inhibition is calculated using the following equation:
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = (1 − (𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑠𝑝𝑖𝑘𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒/(𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑢𝑛𝑠𝑝𝑖𝑘𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 + 𝑆𝐴𝑅𝑆𝑁1 𝑖𝑛 𝑠𝑝𝑖𝑘𝑒𝑑 𝑁𝑇𝐶))) ∗ 100%
Materials
Consumable
  • qScript XLT 1-Step RT-qPCR ToughMix, 20 mL (VWR, cat. no. 95132-02K), aliquots stored at -20°C
  • Fluorescein 10 μM aliquot, stored at −20 °C
  • Nuclease free water, part of Wizard® Enviro TNA Kit (Promega: Cat. No. A2991), aliquots stored at -20°C
  • Primers working solution 10μM, stored at −20 °C
  • Probes working solution 20μM, stored at −20 °C
  • Premixed N2 primers and probe from the SARS-CoV-2 RUO qPCR Primer 26 Probe Kit (IDT: cat. 10006713)
  • Positive control for Resp6 containing at least 30 genome copies of each of the targets (SARS-N1, SARS-N2, IAV, IBV, MHV) (see protocol: Preparation positive controls RESP6 and Inhibition)
  • Inhibition spike aliquot (see protocol: Preparation positive controls RESP6 and Inhibition)
  • 1.5 mL Eppendorf Safe-Lock tubes (Eppendorf, cat. no. 022363212)
  • 2 mL Eppendorf Safe-Lock tubes (Eppendorf, cat. no. 0030120094)
  • 0.5 mL Axygen® Thin Wall PCR Tubes with Flat Cap, Clear, Nonsterile (Corning cat. no. PCR-05-C)
  • Stilla® Sapphire chips (Stilla Technologies, cat. no. C14012)
  • RNase AWAY™ Surface Decontaminant, (Thermo scientific, cat. no. 10666421)
  • 20 μL, 200 μL and 1000 μL GPS LTS pipette filter tips (Mettler Toledo: 30389274, 30389276, 30389293)
  • 200 μL multistep Combitips® advanced Quality (Huberlab, cat. no. 11.4990.21)
  • 1.0 mL multistep Combitips® advanced Quality (Huberlab, cat. no. 11.4990.23)
  • Gloves
  • Lab coat

Equipment
  • Pipets 1000 µL, 200 µL, 20 µL, 10 µL
  • Multistep pipet
  • Vortex mixer (Vortex Genie 2)
  • Table centrifuge (LLG-uniCFUGE 2 Mini Centrifuge)
  • 2 PCR boxes (Mastermix and Template) provided with UV inactivation (Grant bio UVC/T-M-AR PCR cabinet)
  • dPCR geode (Naica‱ system for Crystal Digital PCR‱)
  • dPCR chip reader Prism6 (Naica‱ system for Crystal Digital PCR‱) with integrated computer
  • Antistatic wrist bands and antistatic mats
  • 4 °C refrigerator
  • -80 °C freezer
Analytical material
  • This protocol uses 3x or 5x diluted total nucleic acid extracts from wastewater. Ideally, samples are stored at 4 °C until use in this protocol to prevent RNA degradation by freeze-thawing. However, the protocol can also be used on extracts that have been stored at –80 °C. If frozen at –80 °C, thaw at room temperature, vortex briefly and spin down.
Safety warnings
When using RNAse Away (Thermo Scientific), please consult the manufacturer's instructions and Material Safety Data Sheet
Scope
The goal of this protocol is to determine the PCR inhibition of the RESP6 assay, to ensure that the impact of PCR inhibition on the quantification of viruses stays below the threshold of 40%. Meaning that no more than 40% of the estimate total concentration is not detected due to inhibitory substances in the sample.
Preparation
Prepare an Eppendorf tube (1.5 or 2.0 mL depending on total volume) for the ‘Inhibition mastermix’ and place it opened in a tube rack. Switch on the UV light for 20 minutes.
Prepare 0.5 mL PCR tubes with sample names (1 replicate per samples and at least 2 negative controls per Geode) and place them open in a tube rack.
Take a nuclease-free water aliquot, primer and probe mixes, N2 premix, Q-script, fluorescein out of the 20 °C freezer to let them thaw at room temperature until ice pellets are not longer present.
Mastermix preparation
Vortex and spin down the thawed reagents and aliquots. Take one of the Inhibition spike aliquots, let it thaw, vortex and spin it down.
Prepare the PCR Mastermix according to Table 1.

ReagentVolume per sample (µL)
qScript13.75
Nuclease-free water4.11
N2 premix2.04
Primer mix (10µM) 1.375
Probe mix (20µM)0.275
Fluorescein (10µM)0.138
Inhibition spike (SARS-CoV-2)Dependent on spike quantification
Total volume (µL) per sample22
Table 1: Mastermix composition of reagents and associated volumes per sample for the inhibition Resp6 assay.

Vortex and spin down the mastermix.
Recommendation: Avoid multiple freeze thawing by preparing in advance multiple small volume aliquots. note each cycle of freeze/thaw on the container, as freeze/thaw can reduce efficacy of the constituents.
Addition of template and control
Dispense 22 µL mastermix into each 0.5 mL PCR tube.
Take the samples out of the fridge (or thaw them at room temperature when taking them out of the –80 °C freezer), vortex and spin down.
Recommendation: to help preserve the extracts, the extracts can be put on ice while pipetting.
Pipette 5.5 µL of nuclease-free water into all NTC tubes for negative controls. Change tips for every tube and close them immediately.
According to the labels on the 0.5 mL PCR tubes, pipette 5.5 µL of the extract into the tubes. Caution: change pipet tips each time and prevent sample cross-contamination by closing the tubes once they contain sample.
Put the extracts back in the 4 °C fridge until storage in the –80 °C freezer.
Vortex and spin down all 0.5 mL PCR tubes.
Running the assay
Out of each 0.5 mL PCR tube, take 25 µL and load it onto the labelled Sapphire chips. Loading is done by taking of the flat white cap and adding the sample into the wells by pipetting to the inner wall of the chamber. Afterwards, close the holes with the provided narrow white caps. Caution: it is very important to prevent bubbles while loading chips. Therefore, stop pushing after the ‘first stop’ of the pipet. If there happens to be a bubble at the top, it can be popped with a clean pipet tip.
When all chips are loaded and closed, carefully place the chips in the dPCR geode(s). Make sure to never tilt the chips in any direction.
Close the lid of the geode(s).
Ensure the pressure pump is on and has reached ~0.12 mbar.
Select the program ‘RESPV6' (Table 2) and start the thermocycling by pressing the button with the start triangle.

ABC
Partitioning12 minutes40°C
Reverse Trascription1 hour50°C
Denaturation5 minutes95°C
Thermocycling - 40 cyclesDenature - 30 seconds95°C
Anneal - 1 minute57.5°C
Release pressure

Confirm that you are running Sapphire chips. The Geode is starting now.
Scanning the chips
Normally, the scanning software opens automatically after starting the Prism6 computer and logging into your account. If not, open CrystalReader software.
Click on “New experiment” and select the .ncx file
Adjust the number of geodes to scan using the edit button that looks like a pen in the bottom. Chips or chambers can be added or deleted by clicking the x or + in the corners
Under ‘Experiment details’, click ‘Load sampling names’. Select the template .ncx file provided by stilla technologies.
Click ‘Save as’ and save the file in your computer.
Once the thermocycler program on the geode(s) has finished, put on the antistatic wrist band to take out the chips. Make sure it has contact with your skin and not the glove. Caution: It is now very important to prevent static interaction with the chips. Therefore, ensure you are standing on the antistatic mat and wearing the antistatic wrist band. Also, always first touch the surface where the chips are on, before touching the chips, and always touch a surface you want to place the chips on first as well.
Fill out all ChipIDs in the CrystalReader software by scanning the barcode in the bottom of the Sapphire chips using the barcode scanner connected to the desktop.
Click ‘Open tray’, place the chips in the tray of the Prism6 (three chips at a time). Before placing them, make sure to carefully wipe the dust off with a tissue. Make sure to always keep the chips even and never tilt in any direction when moving them. Click ‘Close tray’.
Click ‘Scan’ to start the imaging. A geode (set of 3 chips) typically takes about 45 min to scan.
When scanning chips from multiple geodes, click the ‘Rescan’ button after placing the next batch of chips in, to start imaging that next batch.
Once imaging is finished, the .ncx file will save automatically. A pop-up window will appear as soon as the saving is finished.
Result processing
If needed, organize samples according to your workflow.
Go to “Setup”, then “Edit Experiment” tab.
Sort the samples by sample name by double clicking the sample name column header.
Go through the list of sample names in the “Edit Experiment” tab and check each input to make sure they are correct.
Quality control of chambers
Go to the ‘Quality control’ tab and go through all chambers. A green square indicates the chamber has passed the crystal miner quality control, an orange one indicates a failed chamber. Reasons for failed chambers can be:
- Less than 12,000 analysable droplets (those are excluded by the wise database automatically after upload)
-Unsharp/unfocused chamber picture (try to replace the chip in the holder and rescan)
- High number of saturated artifacts (carefully clean the bottom with a tissue and rescan)
Go through all the chambers to see if there are any odd droplets. Exclude areas where chips have bubbles or irregular patterns by holding Control, then right click to draw corners of the polygon. Close the polygon by not pressing Control anymore and click one last time. The highest priority for excluding areas is where:
- Positive droplets are not randomly dispersed and clustered together.
- Droplets are slightly larger than surrounding droplets and the software hasn’t recognized it as an artifact.
- Areas around bigger artefacts if they were not excluded automatically.
Gating
Go to ‘Analyze data’ ‘Plots 6 Populations’ and then 2D dot plot.
Change the ‘Type’ to ‘Lines’ and check that the ‘Definition scope’ is set to ‘Common for all chambers’.
Set the axis of each dot plot by double clicking one of the axis labels. Change the first plot to blue y-axis and teal x-axis.
Set the threshold below the droplet clusters. Adjust the naming of populations by clicking on ‘Population Editor’.
Quality control of gating
Go through each sample and monitor that the positive cluster falls above the set threshold. Caution: clusters locations can slightly vary, due to:
- mastermix not being well mixed
- sample to mastermix ratio being off due to too much or too little sample added
- chip being deformed (this needs to be monitored)
- droplet size being slightly larger than usual
- potential sample inhibition
If cluster locations in a sample deviate, examine that chamber in the ‘Quality control’ tab to see if all odd-looking patterns have been excluded.
If an entire geode has clusters in a completely different place, but intra-geode cluster locations are the same, save the entire geode in a separate experiment and analyze it there.
Result export and upload
Go to the ‘Export’ tab and untick all boxes in the list, as stated in the dPCR protocol.
Click ‘Export’.
Quality control
Spiked No Template Controls
At least two NTCs are run per Geode, which use the same mastermix and are therefore spiked with SARS-CoV-2 viral RNA. To calculate inhibition, the average of the concentrations measured in the spiked NTCs is used as the actual number of SARS-N1 genome copies spiked into the mastermix. At least one NTC per inhibition mastermix (can be multiple Geodes) needs to pass quality control in order to calculate the inhibition in the sample.
Inhibition threshold
Inhibition values are calculated using the following equation:
Inhibition = (SARSN1 in spiked sample/(SARSN1 in unspiked sample + SARSN1 in spiked NTC))
- SARS-N1 in spiked sample: the genome copies determined with the inhibition assay in the current protocol.
- SARS-N1 in spiked NTC: the genome copies determined in the NTCs with the inhibition assay in the current protocol.
- SARS-N1 in unspiked sample: the genome copies determined with the RESP6 assay
A threshold of 0.6 is considered acceptable for the RESP6 assay. Values above 0.6 pass inhibition control.
Procedure in case of inhibited samples
Any samples that fail inhibition control must then be further diluted and rerun on both the RESP6 and inhibition assays. Typically, samples are diluted 3x, with the next higher dilution being 5x.
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: Darine D'Adam, Eawag, Swiss Federal Institute of Aquatic Science and Technology