Mar 16, 2026

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

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8xk68v2w/v1
  • 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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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8xk68v2w/v1
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 Six-plex Digital PCR Assay targeting SARS-CoV-2 (N1 and N2), MHV, IAV, IBV, and RSV. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xk68v2w/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: February 16, 2026
Last Modified: March 16, 2026
Protocol Integer ID: 243376
Keywords: digital PCR, Stilla Technologies, wastewater, wastewater monitoring, multiplex, resp6 assay, respiratory viruses sar, plex digital pcr assay, digital pcr assay, wastewater rna extract, respiratory syncytial virus, recovery efficiency control murine hepatitis virus, raw wastewater sample, targeting sar, amount of mhv, resp6, rsv, assay
Funders Acknowledgements:
Swiss National Science Foundation
Grant ID: CRSII5_205933
Swiss National Science Foundation
Grant ID: 142006655/334.0-107/12
Abstract
This protocol describes the procedure for quantification of the respiratory viruses SARS-CoV-2 (N1 and N2 genes), Influenza A (IAV), Influenza B (IBV), Respiratory Syncytial Virus (RSV) and the recovery efficiency control Murine Hepatitis Virus (MHV) in wastewater RNA extracts, using a six-plex digital PCR Assay, heretofore referred to as the Resp6 assay. The assay was developed within the context of Pitton et al. (2024) DOI: 10.1038/s44221-025-00503-x. Extraction efficiency is determined by spiking a known amount of MHV into raw wastewater samples and quantifying the amount extracted during the process.
Materials
Consumables
  • qScript XLT 1-step RT-qPCR ToughMix (QuantaBio: cat. 95132)
  • Fluorescein sodium salt, high purity grade (VWR, cat. no. 0681-100G); aliquot of 10 µM
  • Nuclease-free water (Promega: cat. P1195); aliquots stored at –20 °C
  • Primer mixes for SARS-N1, RSV, IAV, IBV, MHV (Table 2); aliquot with each primer at 10 µM
  • Probe mixes for SARS-N1, RSV, IAV, IBV, MHV (Table 2); aliquot with each probe at 20 µM
  • Premixed N2 primers and probe from the SARS-CoV-2 RUO qPCR Primer & Probe Kit (IDT: cat. 10006713, 5’ Mod.: FAM, Inner mod.: Zen, 3’ Mod.: BHQ11)
  • 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)
  • 1.5 mL or 2.0 mL Eppendorf tubes (Huberlab: cat. 11.3829.02 / 11.3829.03)
  • 0.5 mL PCR tubes (Chemie Brunschwig: cat. AXYPCR-05-C)
  • PCR-Tubes, 8-strips (attached single caps, standard profile, capacity 0.2 mL) (BRAND, cat. Nr. 781332)
  • Stilla‱ Sapphire chips (Stilla Technologies, cat. no. C14012)
  • RNAse Away (VWR: cat. 732-2271)
  • 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‱)
  • Antistatic wrist bands and antistatic mats
  • Computer with CrystalReader and CrystalMiner software (Stilla Technologies)
  • 4 °C refrigerator
  • -80 °C freezer

Analytical material
  • This protocol uses 3x or 5x diluted total nucleic acid extracts from wastewater, prepared as described in LP006. Ideally, samples are stored at 4 °C until used 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.

NameSequenceModification 5'Inner modificationModification 3'
RSV-6p-FCTC CAG AAT AYA GGC ATG AYT CTC C
RSV-6p-RGCY CTY CTA ATY ACW GCT GTA AGA C
RSV-6p-PTAA CCA AAT TAG CAG CAG GAG ATA GAT CAGROX3IAbRQsp - 3' Iowa Black™ RQ-Sp
IAV-6p-FTGG AAT GGC TAA AGA CAA GAC CAA T
IAV-6p-RAAA GCG TCT ACG CTG CAG TCC
IAV-6p-PTTT GTK TTC ACG CTC ACC GTG CCCCy5TAOIowaBlack
IBV-6p-FGAG ACA CAA TTG CCT ACY TGC TT
IBV-6p-RATT CTT TCC CAC CRA ACC AAC A
IBV-6p-PAGA AGA TGG AGA AGG CAA AGC AGA ACT AGCHEXZENBHQ-1
SARSN1-6p-FGAC CCC AAA ATC AGC GAA AT
SARSN1-6p-RTCT GGT TAC TGC CAG TTG AAT CTG
SARSN1-6p-PACC CCG CAT TAC GTT TGG TGG ACC ATTO425ZENBHQ-1
MHV-6p-FGGA ACT TCT CGT TGG GCA TTA TAC T
MHV-6p-RACC ACA AGA TTA TCA TTT TCA CAA CAT A
MHV-6p-PACA TGC TAC GGC TCG TGT AAC CGA ACT GTCy5.53IAbRQsp - 3' Iowa Black™ RQ-Sp
SARSN2N2 mix from SARS-CoV-2 RUO qPCR Primer & Probe Kit (IDT; cat. 10006713)FAM (probe)ZENBHQ-1
Table 2. Primer and probe sequences

Troubleshooting
Safety warnings
When using RNAse Away (Thermo Scientific), please consult the manufacturer's instructions and Material Safety Data Sheet
Scope
This protocol can be used on nucleic acid extracts, including extracts obtained from total nucleic acid extraction from wastewater samples. MHV is spiked into a subset of the samples prior to extraction, and PCR inhibitors are removed post-extraction. This protocol specifically describes the use of the RESP6 assay on the Stilla Naica‱ system for Crystal Digital PCR‱, targeting SARS-CoV-2 N1 and N2 genes, IAV M-gene, IBV M-gene, RSV N-gene and MHV.
The goal of this procedure is to quantify the respiratory viruses SARS-CoV-2 (N1 and N2 genes), IAV M-gene, IBV M-gene, RSV N-gene in wastewater extracts, as well as to quantify the extraction efficiency control MHV in the extracts to calculate the MHV recovery.
Preparation
Clean all relevant laboratory surfaces with an appropriate decontamination reagent, such as RNAse Away (Thermo Scientific) following manufacturer's instructions. Be aware of the surface material types RNAse Away is compatible with. Afterwards, make sure to dry it off with tissues to avoid accumulation of residue on surfaces or equipment.
Label an Eppendorf tube (1.5 or 2.0 mL depending on total volume) as ‘Mastermix’ and place it opened in a tube rack in a dedicated PCR workstation with UV. Then label 0.5 mL PCR tubes or 8-stripe 0.2 mL PCR tubes and place them in a tube rack in the workstation and switch on the UV light for 20 minutes. The number of tubes labeled should be sufficient to include all planned samples including replicates, as well as positive and negative controls (i.e., no template controls or NTCs).
Take a nuclease-free water aliquot, primer and probe mixes, N2 premix, Q-script, fluorescein out of the –20 °C freezer and thaw at room temperature until ice pellets are no longer present in the aliquots.
Label the Stilla‱ Sapphire chips (Stilla Technologies).
Mastermix preparation
Vortex the primer and probe mixes, N2 premix, Q-script (QuantaBio) and fluorescein and then spin down using the tabletop centrifuge.
Prepare the dPCR Mastermix by pipetting the reagents together into the Eppendorf tube, following the volumes per sample as listed in Table 1. Always prepare Mastermix for 2-5 samples extra, to allow for spare volume in the multistep pipet.
AB
ReagentVolume (µL) per sample
Q-script MasterMix13.75
Nuclease-free water4.43
N2 premix2.04
Primer mix SARS-N1/IAV/IBVR/SV/MHV (10 µM)1.375
Probe mix SARS-N1/IAV/IBV/RSV/MHV (20 µM)0.275
Fluorescein (10 µM) 0.137
Total volume (µL) per sample22
Table 1: Mastermix composition of reagents and associated volumes per sample for the Resp6 assay.

Vortex the Mastermix and spin it down.
Return unused primer and probe mixes, N2 premix, Q-script and fluorescein back to the -20 °C freezer.
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 controls
Dispense 22 µL Mastermix into each 0.5 mL PCR tube.
Take the RESP6 positive control out of the –80 °C freezer and let it thaw at room temperature. Once thawed, vortex and spin down. The RESP6 positive control preparation is described elsewhere. Concentrations of each target should be sufficient to observe not only single positive droplets but also double positive droplets..
Take the samples out of the fridge or thaw at room temperature if previously frozen, vortex and spin down. Recommendation: to help preserve the samples, the samples and positive control can be put on ice while pipetting.
Recommendation: freeze-thaw cycles degrade nucleic acids and should be avoided. For long term storage, samples stored at -80C are typically more stable than samples stored at -20C or +4C.
According to the labels on the 0.5 mL PCR tubes or 0.2 mL PCR 8-strips, pipette 5.5 µL of that samples into the tube. Caution: change pipet tip each time and prevent sample cross-contamination by closing the tubes once they contain sample or control.
Pipette 5.5 µL of the RESP6 positive control into the designated tubes. Throw aliquot away after use.
Pipette 5.5 µL of nuclease-free water into the tubes for negative controls. Throw aliquot away after use.
Put the samples back in the 4 °C fridge.
Vortex and spin down all 0.5 mL PCR tubes or 0.2 mL PCR 8-strips. Caution: the volume in those tubes is very low. To ensure good mixture, either vortex them few seconds, or spin the tubes before vortexing, then vortex and spin again.
Running the assay
Out of each 0.5 mL PCR tube or 0.2mL PCR 8-strip tube, take 25 µL and load it onto the labelled Sapphire chips. Loading is done by taking off the flat white cap and pipetting the sample into the holes. Make sure to pipet to the wall of the chamber. Afterwards, close the holes with the provided narrow white caps.
When all chips are loaded and closed, carefully place the chips in the dPCR geode(s). Make sure to always keep the chips horizontally and do not tilt them.
Close the lid of the geode(s).
Ensure the pressure pump is on and has reached ~0.12 mbar.
Store the program RESP6 in the memory of the geode, select the program from the menu, and start the thermocycling (program in Table 3).
ABC
Partitioning12 minutes40°C
Reverse Transcription1 hour50°C
Denaturation5 minutes95°C
Thermocycling - 40 cyclesDenature - 30 seconds95°C
Anneal - 1 minute57.5°C
Release pressure
Table 3. RESP6 thermocycling program

When program is running, store the remainder of the samples in the -80C freezer and note if they have undergone freeze / thawing.
Warning: Multiple freeze/thaw cycles can reduce nucleic acid concentrations. Avoid multiple cycles by aliquoting small volumes of samples prior to freezing.
Scanning the chips
Normally, the scanning software opens automatically when starting the Prism6. If not, open CrystalReader software on the Prism6.
Click on “New experiment” and select the template .ncx file provided by Stilla Technologies.
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.
Click ‘Save as’ and save the file in your project's data directory
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.
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 (one geode at a time). Before placing the chips, make sure to carefully wipe the bottom of the chips with a tissue to remove dust or other artifacts that might interfere with imaging. Make sure to always keep the chips upright even when moving them. Click ‘Close tray’.
Click ‘Scan’ to start the imaging.
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
Open the .ncx file and organize the samples by geode
Once the ncx file is saved, click ‘Open in CrystalMiner’ to start the analysis. If CrystalReader was closed in the meantime, double click on the ncx file and it will open a ncr file in CrystalMiner automatically.
To add data from a different experiment, click on “add chambers” and select the respective ncx file.
Organize the samples by geode as follows:
- 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 raw images (manual exclusion of droplets)
Go to the ‘Quality control’ tab and review all chambers. Chambers with an orange square have automatically failed quality control (often because they contain ≤ 12,000 analyzable droplets). Although Stilla Technologies officially recommends ≥ 15,000 droplets per chamber, we have observed that ~12,000 analyzable droplets can still yield reliable results. If both technical replicates of a sample have failed, the sample will need to be repeated.
Go through all the chambers to see if there are any irregular droplets. Exclude droplets 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 is excluding areas where:
- Positive droplets are not randomly dispersed.
- 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
In the CrystalMiner software, go to ‘Analyze data’ ‘Plots & Populations’ and then 2D dot plot.

Change the ‘Type’ to ‘Lines’ and check that the ‘Definition scope’ is set to ‘Common for all chambers’.
Select all positive controls on the left-hand side tab. Set the axis of each dot plot by double clicking one of the axis labels (ie. Yellow or Red).
- Change the first plot to blue y-axis and teal x-axis.
- The second plot to yellow y-axis and red x-axis.
- The third plot to infra-red y-axis, and red x-axis.
- The fourth plot to blue y-axis and green x-axis.
With only positive controls selected, set the threshold lines for each target. Use colors shown below as they help the next user easily identify which target is on which plot. Adjust the naming of populations by clicking on ‘Population Editor’.
Thresholds should be set in a way that they cut close to the positive cluster.
Check for contamination in NTCs
After all the thresholds have been set, go through each no template control (NTC) one by one and check the number of positive droplets above the threshold.
If there are 3 or more positive droplets the geode is considered contaminated and all samples on the geode need to be repeated.
Recommendation: The limit of detection of an assay is determined by the limit of blank, which we set to 2 or fewer positive droplets. This threshold may vary by laboratory and assay, but should be informed by multiple replicates of No Template Controls.
If an NTC has not enough total droplets (failed quality), but has less than three positive droplets, the geode will pass as long as no other geodes run from the same master mix have a contaminated NTC.
Quality control of clusters
Go through each sample and monitor that the positive cluster falls above the threshold line. This can be easily checked in the 1D plot.
If cluster locations in a sample deviate, examine that chamber in the ‘Quality control’ tab to see if all irregular patterns have been excluded.
If both replicates of a sample show cluster shifts below the threshold, the sample will need to be repeated.
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 exporting
Go to the ‘Export’ tab and tick boxes for files you want to export.
Click ‘Export’.
Quality control
Per geode, we recommend using a positive control (POS) and negative control (NTC). Positive controls contain nucleic acid material for all targets in the assay. For the negative controls, nuclease-free water is added to the Mastermix, instead of sample. The sample on the geode are deemed non-contaminated if there are less than 3 positive droplets for all targets in the negative control.
Recommendation: Stilla Technology Sapphire chips have a manufacturer failure rate of insufficient analyzable droplets of ~3-5%. The threshold of <= 12.000 analyzable droplets can be reconsidered based on desired performance of quantification estimates and the associated uncertainty or variability. When positive or negative controls fail due to insufficient analyzable droplets, care should be taken in differentiating manufacturer failures from contamination events.
Sample check with MHV quantification
In this protocol, MHV is quantified to determine the amount of spiked-in MHV that was recovered during total nucleic acid extraction.
MHV quantification also serves as a quality control measure to verify the correct sample order, as every other sample should contain spiked MHV. If the MHV concentration does not follow the expected pattern, the sample extraction and/or dPCR analysis will be repeated.
If three or more MHV-positive droplets are found in an unspiked sample, contamination may have occurred. First, the dPCR needs to be repeated for this sample, as well as for the spiked samples directly preceding and following it. If contamination continues after these repeats, perform re-extraction for the contaminated samples as well as the samples before and after the affected one in the process order.
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