Jun 02, 2026
Version 1
RESPV6 Respiratory virus quantification on Bio-Rad QX600 Digital Droplet PCR System V.1
- Melissa Pitton1,
- Timothy R. Julian1,2,3,
- Christoph Ort1
- 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland;
- 2Wastewater Monitoring Laboratory;
- 3Eawag Aquatic Research
Protocol Citation: Melissa Pitton, Timothy R. Julian, Christoph Ort 2026. RESPV6 Respiratory virus quantification on Bio-Rad QX600 Digital Droplet PCR System. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqxx5klk5/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: May 12, 2026
Last Modified: June 02, 2026
Protocol Integer ID: 316866
Keywords: respv6 respiratory virus quantification on bio, respv6 respiratory virus quantification, respv6 assay, recovery efficiency control murine hepatitis virus, respiratory viruses sar, respiratory syncytial virus, digital pcr assay, raw wastewater sample, plex digital pcr assay, rad qx600 digital droplet pcr system, rad qx600 digital droplet pcr system this protocol, qx600 system from bio, wastewater extract, qx600 system, amount of mhv, extraction efficiency, rsv, extraction
Abstract
This protocol describes the procedure for quantification of the respiratory viruses SARS-CoV-2 (N1 and N2 targets), Influenza A (IAV), Influenza B (IBV), Respiratory Syncytial Virus (RSV) and the recovery efficiency control Murine Hepatitis Virus (MHV) in wastewater extracts, using a six-plex digital PCR assay (RESPV6 assay) on the QX600 system from Bio-Rad. Extraction efficiency is determined by spiking a known amount of MHV into raw wastewater samples and quantifying the amount that got extracted during the process.
Image Attribution
Image sourced from the Bio-Rad AutoDG user manual.
Materials
- One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad: cat. no. 1864022) that contains three individual tubes: Supermix, Reverse transcriptase (RT), 300 mM DTT
- Automated Droplet Generation Oil for Probes
- ddPCR™ Droplet Reader Oil
- ddPCR™ Buffer Control for Probes
- RNase away
- Nuclease-free water (Promega: cat. no. P1195); aliquots stored at -20°C
- Primer mixes (see Table below), aliquot with each primer at 7.2 µM
- Probe mixes (see Table below), aliquot with each probe at 2 µM
- Positive control for RESPV6
- 1.5 mL or 2.0 mL Eppendorf tubes
- Automated Droplet Generation Oil for Probes
- Pipet Tips for the AutoDG™ System
- DG32™ Automated Droplet Generator Cartridges (Bio-Rad: cat. no. 1864109)
- Pipet™ Waste Bins for the AutoDG™ System (Bio-Rad: cat. no. 1864125)
- ddPCR™ Droplet Reader Oil (Bio-Rad: cat. no 1863004)
- ddPCR™ Buffer Control for Probes (Bio-Rad: cat. no 1863052)
- ddPCR™ 96-Well Plates (Bio-Rad: cat. no. 12001925)
- PCR Plate Heat Seal, foil, pierceable (Bio-Rad: cat. no. 1814040)
- RNase Away
- 20 µL, 200 µL and 1000 µL pipette filter tips
- 10 mL multistep Combitips advanced Quality
- Gloves
- Lab coat
- Protective glasses
- Permanent marker
- Table centrifuge
- 2 PCR boxes (Mastermix and Template)
- QX600 AutoDG Droplet Digital PCR System (Bio-Rad) (includes reader and droplet generator)
- PX1 PCR Plate Sealer (Bio-Rad)
- Computer with QX Manager software (Bio-Rad)
- 4°C refrigerator
- -80°C freezer
- CVP-2, Centrifuge-Vortex for PCR plates (Biosan)
- PTC Tempo Thermal Cycler
- Eppendorf® PCR cooler, 0.2 mL
| Name | Sequence (5’-3’) | Modification | |
| RSV-6p-F | CTC CAG AAT AYA GGC ATG AYT CTC C | ||
| RSV-6p-R | GCY CTY CTA ATY ACW GCT GTA AGA C | ||
| RSV-6p-P | TAA CCA AAT TAG CAG CAG GAG ATA GAT CAG | 5'-/56-ROXN/TAA CCA AAT TAG CAG CAG GAG ATA GAT CAG /3IAbRQSp/-3' | |
| IAV-6p-F | TGG AAT GGC TAA AGA CAA GAC CAA T | ||
| IAV-6p-R | AAA GCG TCT ACG CTG CAG TCC | ||
| IAV-6p-P | TTT GTK TTC ACG CTC ACC GTG CCC | 5'-/5Cy5/TTT GTK TTC /TAO/ ACG CTC ACC GTG CCC /3IAbRQSp/-3' | |
| IBV-6p-F | GAG ACA CAA TTG CCT ACY TGC TT | ||
| IBV-6p-R | ATT CTT TCC CAC CRA ACC AAC A | ||
| IBV-6p-P | AGA AGA TGG AGA AGG CAA AGC AGA ACT AGC | 5'-/5HEX/AGA AGA TGG /ZEN/ AGA AGG CAA AGC AGA ACT AGC /3IABkFQ/-3' | |
| SARSN1-6p-F | GAC CCC AAA ATC AGC GAA AT | ||
| SARSN1-6p-R | TCT GGT TAC TGC CAG TTG AAT CTG | ||
| SARSN1-6p-P | ACC CCG CAT TAC GTT TGG TGG ACC | 5'-/5ATTO590N/ACC CCG CAT TAC GTT TGG TGG ACC /3IAbRQSp/-3' | |
| MHV-6p-F | GGA ACT TCT CGT TGG GCA TTA TAC T | ||
| MHV-6p-R | ACC ACA AGA TTA TCA TTT TCA CAA CAT A | ||
| MHV-6p-P | ACA TGC TAC GGC TCG TGT AAC CGA ACT GT | 5'-/5Cy55/ACA TGC TAC GGC TCG TGT AAC CGA ACT GT /3IAbRQSp/-3' | |
| SARSN2-6p-F | TTA CAA ACA TTG GCC GCA AA | ||
| SARSN2-6p-R | GCG CGA CAT TCC GAA GAA | ||
| SARSN2-6p-P | ACA ATT TGC CCC CAG CGC TTC AG | 5'-/56-FAM/ACA ATT TGC /ZEN/ CCC CAG CGC TTC AG /3IABkFQ/-3' |
Safety warnings
Please make sure you are aware of the safety data sheets and hazards of the following consumables:
- One-Step RT-ddPCR Advanced Kit for Probes
- Automated Droplet Generation Oil for Probes
- ddPCR™ Droplet Reader Oil
- ddPCR™ Buffer Control for Probes
- RNase away
Caution: Remove plates from AutoDG as quickly as possible ( 30 min) to avoid degradation of reagents and/or target of interest.
Scope and Goal
This protocol can be used on wastewater extracts, obtained by extracting total nucleic acids from raw wastewater samples, where MHV is spiked into part of the samples pre-extraction and PCR inhibitors are removed post-extraction.
This protocol specifically describes the use of the RESPV6 assay on the Bio-Rad QX600 Digital Droplet PCR System, 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, IBV and RSV in wastewater extracts, as well as to quantify the extraction efficiency control MHV in the extracts to calculate the MHV recovery.
Preparation
Important: The QX600 AutoDG ddPCR System operates in an 8-well column format. If fewer than 8 wells are used within a column, the remaining wells must be filled with Buffer Control. This requirement does not apply to columns that are entirely unused.
Clean the PCR mastermix and template boxes with RNAse away. Afterwards, make sure to dry it off with tissues to avoid accumulation of residue on surfaces or equipment.
Label an small centrifuge tube (1.5 or 2.0 mL depending on total volume) as “Mastermix” and place it opened in a tube rack in the PCR Mastermix box.
Switch on the UV light for 20 minutes.
Place two 96-well plates in the PCR Template box, as for every 96-well plate needed, an additional plate needs to be sterilized (droplet generation).
Place the multistep pipet, multichannel pipet, and pipet tips in the PCR Template box and switch on the UV light for 20 minutes.
In the meantime, take a nuclease-free water aliquot, primer and probe mixes, supermix, reverse transcriptase, and DTT out of the -20°C freezer to let them thaw at room temperature.
Mastermix preparation
Vortex thoroughly for 30 sec and spin down the primer and probe mixes, supermix, reverse transcriptase, and DTT.
Work in the PCR Mastermix box.
Prepare the Mastermix according to the manufacturer's instructions by pipetting the reagents together into the centrifuge tube, following the volumes per sample as listed in table 1.
| Reagent | Volume (µL) per sample | |
| Supermix | 5.60 | |
| Reverse Transcriptase | 2.24 | |
| DTT (300 mM) | 1.12 | |
| Nuclease-free water | 2.24 | |
| Primer mix (7.2 µM) | 2.80 | |
| Probe mix (2 µM) | 2.80 | |
| Total volume (µL) | 16.80 |
Table 1: Mix of reagents for RESPV6 Mastermix per sample.
Vortex the Mastermix and spin it down. Place it in the PCR Template box.
Put all the Mastermix reagents back in the -20°C freezer.
Empty the waste bin, clean the PCR Mastermix box with appropriate cleaning products such as RNAse Away and switch on the UV light for 20 minutes.
Addition of template and controls
Work in the PCR Template box.
Dispense 16.8 µL Mastermix into each well using the multistep pipette.
Ensure the 96 well plate is on a centrifuge tube PCR cooler if more than ¼ of the plate is being pipetted at a time to avoid spending too much time at room temperature for the reagents.
Take the RESPV6 positive control out of the -20°C freezer and let it thaw at room temperature. Once thawed, vortex and spin down.
Take the wastewater extracts 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 and positive control can be put on ice while pipetting.
Using the pipette transfer 5.6 µL of wastewater extract into the correct well.
Caution: change pipet tip for each sample.
Recommendation: arrange the wastewater extracts on a rack in the correct order for pipetting.
Pipette 5.6 µL of the RESPV6 positive control into the designated wells.
Pipette 5.6 µL of nuclease-free water into the wells for no-template controls. Throw aliquot away after use.
Put the wastewater extracts back in the 4°C fridge.
If fewer than 8 wells are used within a column, add 22.4 µL of 1x Buffer Control in the remaining wells.
Place an aluminium foil sheet on top of the plate.
Caution: Ensure the red line is visible and positioned on the upper side of the plate.
Remove the plate from the PCR Template box.
Empty the waste bin, clean the PCR Template box with appropriate cleaner, such as RNAse Away, and switch on the UV light for 20 minutes.
Plate sealing and vortexing
Turn on the plate sealer, remove the metal block, and allow the unit to warm up to 180°C.
Place the plate on the metal block and seal the plate (180°C for 5 s).
Caution: Ensure that the red line on the aluminium foil is visible and positioned on the upper side of the plate.
After sealing, remove the metal block from the sealer again.
Vortex and centrifuge (1 min at 1’000 RCF) the sealed plate, for example using a CVP-2, Centrifuge-Vortex for PCR plates with the SMS program, “Spin Mix Spin”)
Check visually for bubbles.
Caution: If many small bubbles are visible, repeat previous step. If a single large bubble is present, it should not be an issue, as it will be eliminated during the AutoDG run.
Plate Loading Procedure for AutoDG Droplet Generation
Ensure that the correct oil is installed (“Automated Droplet Generation Oil for Probes”)
Remove the plastic wrapping from cartridges and their white rack and place them in the correct position.
Caution: The green side should be on the right-hand side.
Load AutoDG pipet box(es) by removing the plastic wrapping and removing its lid.
Caution: For one column on the plate, you need two columns of pipet tips.
Place the sealed plate containing your PCR reactions in the position “Sample plate” and check for correct orientation (i.e., well A1 positioned at the top-left corner).
Get a cooling block (stored upside down) from the freezer (-20°C) and place it in the AutoDG in position “Droplet plate”.
Caution: The cooling block should have been at -20°C for at least 2 h prior to run. If it is not sufficiently cool anymore, its color will turn from dark purple to pink.
Place the empty 96-well plate (“Droplet Plate”) in the cooling block (previously sterilized using UV light).
Verify that all required materials have been correctly loaded into the AutoDG system.
Click “Configure Sample Plate” and select the columns that are currently filled (i.e., in use).
Click “Start Run” and keep the lid closed during the droplet generation (this step requires ~45 min for one full 96-well plate).
When program is running, store the wastewater extracts in the -80 °C freezer.
Once droplet generation is complete, remove both 96-well plates. Discard the “Sample Plate” and reseal the “Droplet Plate” (same workflow as described under section "Plate sealing and vortexing").
Caution: Remove plates from AutoDG as quickly as possible (< 30 min) to avoid degradation of reagents and/or target of interest.
Remove the empty pipet tip boxes, trash the cartridges, and trash the tips from the “Tip waste bin”. The “Tip Waste Bin” is sprayed with appropriate disinfectant such as RNAse away and put under UV for 20min after use for decontamination.
Place the cooling block back into the freezer in an upside-down orientation at -20°C.
AutoDG is made to be in standby mode, therefore do not switch off entirely.
Seal your plate with generated droplets using the sealer and aluminium foil.
Thermocycling
Use the PTC Tempo Thermal Cycler (Bio-Rad).
Select the program RESPV6 (Table 2). Important: all steps require a ramp rate of 2°C/second, a lid temperature of 105°C, and a well volume of 40 µL.
| Step | Temperature (°C) | Time | Number of cycles | |
| Reverse transcription | 50 | 60 min | 1 | |
| Enzyme activation | 95 | 10 min | 1 | |
| Denaturation | 95 | 30 sec | 40 | |
| Annealing/Extension | 57.5 | 1 min | 40 | |
| Enzyme Denaturation | 98 | 10 min | 1 | |
| Hold | 4 | 30 min | 1 |
Table 2: RESPV6 Thermocycling Program
Reading Data
Turn on the QX600 reader and the computer connected to it.
Open the latest version of “QX Manager Standard Edition” software.
The reader checks for oil levels and if a plate is inserted. In case oil levels are low, the light blinks green. If oil levels are too low, the light blinks orange and the system prevents you from starting any reads.
Open the reader by clicking on the button on the green lid.
Insert your “Droplet Plate” by using the metallic plate holder (base and lid). Well A1 must be top left. Press down the black tabs on the side to firmly secure the plate in place. Close the reader again.
Make sure the first 3 indicator lights on the QX600 are green.
If the reader is connected to the computer, the top row shows the “Instrument status bar”.
Click on “Add Plate” and then on “Configure Plate”. Insert Plate information
File Name (to do in advance)
Use a name filler to create sample names and then a “Plate template” which can be loaded directly into the QX Manager software. In the QX Manager Standard Edition Interface, click Browse to select and upload the Plate Template. Make sure the “Save data file as” field shows the same name you gave the Plate Template.
Click “Well Selection” and exclude any wells that do not need to be read.
Click “Start Run” to start the reading.
Result exporting
Click on “Data Table”. Click on the "hamburger" icon on the top corner right, and select “Export to CSV.”
Make sure the export will be saved in the desired path so that you will find your data at a later timepoint again.