May 02, 2022
Version 5
High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR V.5
- Aaron Topol (Verily Life Sciences)1,
- marlene.wolfe1,
- Brad White (Verily Life Sciences)1,
- Krista Wigginton1,
- Alexandria B Boehm1
- 1Stanford University
Protocol Citation: Aaron Topol (Verily Life Sciences), marlene.wolfe, Brad White (Verily Life Sciences), Krista Wigginton, Alexandria B Boehm 2022. High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw5orwvmk/v5Version created by Alexandria B Boehm
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 02, 2022
Last Modified: May 02, 2022
Protocol Integer ID: 61803
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Abstract
v3 updates: changes to the S gene primers and probes for detection of all SARS-CoV-2 variants
This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules.
This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses.
This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL).
Scope
This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
Materials
Equipment
●Bio-Rad QX200 AutoDG
●Bio-Rad QX200 Droplet Reader
●Bio-Rad PX1 PCR Plate Sealer (Bio-Rad, Catalog #:1814000)
●Agilent Bravo
●Eppendorf Master Cycler ProThermal Cycler
●Bio-Rad C1000 Touch Thermal Cycler
●Rainin Single-Channel Pipettes L1000XLS+, L200XLS+, L20XLS+, L10XLS+, , L2XLS+
●Rainin Multi-Channel Pipettes L12-1000XLS+, L12-200XLS+, L12-20XLS+, L12-10XLS+, L8-1000XLS+, L8-200XLS+, L8-20XLS+, L8-10XLS+
●Axygen Plate Centrifuge
●Benchtop vortex
●96-well plate cold block for AutoDG droplet generator
●96-well plate cold block for ddPCR plates
●Zebra GX430t label printer
Reagents
●ddPCR™ One-Step RT supermix for Probes
●Reverse Transcriptase
●300mM DTT
●Automated Droplet Generation Oil for Probes
●Ambion Nuclease Free Water, 50mL
●ddPCR Droplet Reader Oil
Consumables
●Bio-Rad ddPCR™ 96-Well Plates
●Eppendorf twin.tec PCR Plate
●DG32™ Automated Droplet Generator Cartridges
●PCR PX1 Plate Sealer, foil, pierceable
●Pipet Tips for the AutoDG™
●Rainin low retention, pre-sterilized, filter tips 1000 µL, 200 µL, 20 µL
●Axygen Automation Tips, 20 µl, filtered, sterile tips (Axygen 20 µL tips)
●Reagent Reservoirs
●Automation Reservoirs
●Eppendorf Tubes, 1.5mL, 2mL and 5mL.
●2.5” x 0.5” Cryogenic Labels for the Zebra GX430t label printer
Samples and Test Materials
●Purified RNA from wastewater samples as described in XXXX
●SARS-CoV-2 gRNA positive control (ATCC® VR-1986D™) diluted and aliquoted at a concentration of 50 copies per µL.
●BCoV and PMMoV gene block positive control diluted and aliquoted at a concentration of 50 copies per µL.
Protocol materials
One-Step RT-ddPCR Advanced Kit for ProbesBio-Rad LaboratoriesCatalog #186-4021
In 2 steps
Preparation (both assays)
Preparation (both assays)
Retrieve all kit components from the One-Step RT-ddPCR advanced kit for probes from the -20 °C freezer and thaw the components On ice .
One-Step RT-ddPCR Advanced Kit for ProbesBio-rad LaboratoriesCatalog #186-4021
Retrieve ddPCR positive control aliquots (50 copies per uL gRNA and 100 copies per µL BCoV and PMMoV gene blocks) from the -80 °C freezer and thaw On ice
For re-running frozen plates only:
Thaw the RNA storage plate On ice . Before analysis of thawed frozen samples, ensure that the plate is adequately sealed and briefly vortex and centrifuge the plate to ensure the samples are well mixed.
Keep extracted RNA samples On ice or in a cold block from the freezer at all times.
Note
IMPORTANT NOTE: The PMMoV assay is very sensitive to contamination due to the high copy number of PMMoV RNA molecules in the lab. It is essential to always use freshly prepared water and other reagents when setting up the BCoV/PMMoV assay and the change gloves and wipe down the working space and pipettes frequently with clean water.
ddPCR master mix preparation (SARS-CoV-2 targets)
ddPCR master mix preparation (SARS-CoV-2 targets)
45m
45m
Prepare SARS-CoV-2 Target Master Mix
Note
This is a triplex assay for the N, S, and ORF1a gene. It is possible to swap out one or two of the assays for others that also target low copy number targets. However, we do not recommend multiplexing with a high copy number target like PMMoV because a high copy number target assay would need to use diluted RNA as template.
Prepare a working stock of fresh nuclease free water by pouring from a 50 mL Ambion Nuclease Free water into a 5mL eppendorf tube.
Store Master Mix components On ice as much as possible during the preparation process.
Briefly vortex One-Step RT supermix for Probes (Yellow Tube), Reverse Transcriptase (Orange Tube), and DTT (Grey Tube) to mix contents then briefly spin with the benchtop centrifuge.
One-Step RT-ddPCR Advanced Kit for ProbesBio-rad LaboratoriesCatalog #186-4021
In a 2mL eppendorf tube, prepare the master mix according to the table for SARS-CoV-2 quantification. Store prepared master mix on ice.
Note
The 20x SARS-CoV-2 Primer/Probe Mix contains primers and probes in the following concentrations (suspended in molecular grade water):
| A | B | C | |
| 20x Concentration | Final Concentration/Rxn | ||
| Primers (each) | 18 µM | 900 nM | |
| N Probe (FAM) | 5 µM | 250 nM | |
| S Probe (HEX) | 5 µM | 250 nM | |
| ORF1a Probe (HEX) | 2.5 µM | 125 nM | |
| ORF1a Probe (FAM) | 2.5 µM | 125 nM |
| A | B | C | |
| Reagents | Volume per Well | Volume Per Plate | |
| ddPCR™ One-Step RT supermix for Probes (Yellow Tube) | 5.5 µL | 580.8 µL | |
| 20x SARS-CoV-2 Primer/Probe Mix | 3.3 µL | 348.48 µL | |
| Reverse Transcriptase (Orange Tube) | 2.2 µL | 232.32 µL | |
| 300mM DTT (Gray Tube) | 1.1 µL | 116.16 µL | |
| Nuclease Free Water | 4.4 µL | 464.64 µL | |
| Total Volume | 16.5 µL | 1742.4 µL |
SARS-CoV-2 ddPCR Master Mix. Volume per plate assumes 96 well plate (with 10% excess).
ddPCR master mix preparation (BCoV and PMMoV)
ddPCR master mix preparation (BCoV and PMMoV)
Prepare Master Mix for the BCoV/PMMoV assay
Note
Store Master Mix components on ice as much as possible during the preparation process.
Briefly vortex One-Step RT supermix for Probes (Yellow Tube) and Reverse Transcriptase (Orange Tube) to mix contents then briefly spin with the benchtop centrifuge. Thaw DTT (grey tube).
In a 2mL eppendorf tube, prepare the master mix according to the table for PMMoV/BCoV quantification. Store prepared master mix on ice.
Note
The 20x BCoV/PMMoV Primer/Probe Mix contains primers and probes in the following concentrations (suspended in molecular grade water):
| A | B | C | |
| 20x Concentration | Final Concentration/Rxn | ||
| Primers (each) | 18 µM | 900 nM | |
| BCoV Probe (FAM) | 5 µM | 250 nM | |
| PMMoV Probe (HEX) | 5 µM | 250 nM |
| A | B | C | D | E | |
| Reagents | Volume per Sample | Volume Per Plate | |||
| ddPCR™ One-Step RT supermix for Probes (Yellow Tube) | 5.5 µL | 580.8 µL | |||
| BCoV/PMMoV Primer/Probe Mix | 2.2 µL | 232.32 µL | |||
| Reverse Transcriptase (Orange Tube) | 2.2 µL | 232.32 µL | |||
| 300mM DTT (Gray Tube) | 1.1 µL | 116.16 µL | |||
| Nuclease Free Water | 5.5 µL | 580.8 µL | |||
| Total Volume | 16.5 µL | 1742.4 µL |
BCoV and PMMoV ddPCR Master Mix. Volume per plate assumes 96 well plate (with 10% excess).
ddPCR plate set up (BCoV and PMMoV)
ddPCR plate set up (BCoV and PMMoV)
Dilute the RNA 1:100 for BCoV/PMMoV RT-ddPCR
Note
The RNA must be diluted because of the high expected concentration of these targets.
Note
The dilution of the RNA can be completed using a liquid handling robot. We use the Agilent Bravo system.
Using a new reagent reservoir, manually pipette 198 µL of nuclease free water into every well of an Eppendorf twin.tec PCR Plate labeled “100x RNA Dilution”.
Manually pipette 2 µL of RNA from the stock RNA plate into each corresponding well in the dilution plate.
Seal the plate and briefly vortex the plate using a bench top plate vortexer.
Briefly spin down the plate in the Axygen Plate Centrifuge.
Store plate On ice until ready to add sample to ddPCR plate.
Transfer Master Mix and Samples to ddPCR plate (both assays)
Transfer Master Mix and Samples to ddPCR plate (both assays)
For each assay, remove a white cold block from the freezer and place a new Bio-Rad ddPCR 96-well Plate in it.
Note
Note that these steps may be completed with a liquid handling robot. We use the BRAVO system.
Using a new reagent reservoir, manually pipette 16.5 µL of the appropriate Master Mix to each well of each ddPCR Plate.
Transfer samples to the following wells on the plate either manually or using a liquid handling robot such as the Agilent Bravo system:
- Transfer 5.5 µL of samples from columns 1-10 of the RNA plate into columns 1-10 of the ddPCR plate.
- Transfer 5.5 µL of the extraction controls from column 12 of the RNA plate into column 11 of the ddPCR plate.
- Transfer 5.5 µL of NTC (water) into A-G of column 12.
- Add 5.5 µL of ddPCR positive control to well H12 of the ddPCR plate (add manually even if using a liquid handler)
Note
For SARS-CoV-2 PCR: Add SARS-CoV-2 genomic RNA
For PMMoV/BCoV PCR: Add PMMoV/BCoV gene block positive control
The plate layout should be:
Note
Note: The set up of this plate is slightly different from the set up of the 96 well extraction plate used in our companion protocol. Column 11 of this plate uses column 12 of the extraction plate. The lower right hand corner was purposely chosen for the placement of the positive controls.
Bring the BioRad ddPCR plate with Master Mix and samples to the Bio-Rad PX1 PCR Plate Sealer and place it in the metal carrier inside the plate sealer.
Note
Do not store the metal carrier inside the plate sealer, as it will become warm and warm the samples during sealing.
Align a PCR Plate Heat Seal Foil on top of the ddPCR plate with the red line facing up.
Seal the plate by pressing the green “seal” button.
Briefly vortex the plate using a bench top plate vortexer.
Briefly spin down the plate in the bench top Axygen Plate Centrifuge.
Store the ddPCR plate on a white cold block or On ice until droplet generation.
ddPCR droplet generation (both assays)
ddPCR droplet generation (both assays)
45m
45m
Generate Droplets:
On the AutoDG droplet generator: select “configure plate” and then press the blue arrow in the upper left corner to highlight all columns.
Load 3 DG32 cartridge plates and 2 tip boxes with the lid removed onto the AutoDG deck. The icons on the AutoDG display will change from yellow to red if the plates and tips are oriented correctly and to green if placed correctly.
Place the sealed, vortexed, and centrifuged ddPCR plate in the Sample Plate position on the AutoDG deck.
Remove the AutoDG cooling block from the freezer and place it in the Droplet Plate slot on the AutoDG deck.
For the Droplet Plate: label a new Bio-Rad ddPCR™ 96-Well Plate and place it in the black cooling block.
Click “Generate Droplets” to begin droplet generation. Droplet generation will take approximately 00:45:00 .
Note
During Droplet Generation for the SARS-CoV-2 plate, set up the PMMoV/BCoV plate.
45m
When droplet generation is done, open the lid and remove the droplet plate from the black cold block and place it in the metal carrier inside the plate sealer.
Align a PCR Plate Heat Seal Foil on top of the droplet plate with the red line facing up.
Photograph showing proper setup of samples and consumables in the AutoDG. After droplet generation, samples have been emulsified and deposited into the "Droplet Plate," which is ready for thermocycling.
Seal the plate by pressing the green “seal” button.
Note
NOTE: Do NOT vortex plate at this point!
Thermocycling (SARS-CoV-2)
Thermocycling (SARS-CoV-2)
45m
45m
Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for SARS-CoV-2 ddPCR.
Cycling conditions for the SARS-CoV-2 RNA target assay
After the PCR program is done, store the ddPCR plate at 4 °C (in the thermal cycler, on ice or in the fridge) until droplet reading.
Sample storage
Sample storage
Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for BCOV/PMMoV ddPCR.
Cycling conditions for the PMMoV / BCoV assay
After the PCR program is done, store the ddPCR plate at 4 °C (in the thermal cycler, on ice or in the fridge) until droplet reading.
Read Droplets (both assays)
Read Droplets (both assays)
Before running the plate on the QX200 droplet reader, open the drawer on the left side of the instrument and check that there is adequate droplet reader oil and that the waste bottle is not overly full.
Load template from previous plate of the same assay - ensure that all parameters (dye, absorbance etc.) are correct for the assay used and that sample names are assigned to the appropriate wells. Save as with a new plate name.
Name the file with a name clearly describing which plate you are reading.
Remove the plate from the thermal cycler and secure it in the droplet reader by placing it in the stage, placing the metal brace on top of it and pressing down the black plastic tabs on either side of the brace.
Click “Run” in Quantasoft to commence droplet reading.
Select “Ok” when the next dialog box comes up. Do not change the settings from “Columns” and “FAM/HEX”.
SARS-CoV-2 Post-Processing Analysis
SARS-CoV-2 Post-Processing Analysis
SARS-CoV-2 triplex assay analysis
Open QuantaSoft™ Analysis Pro Software
Open the .qlp file associated with the run
1.1.1.On the Plate Editor tab, select all the wells then in the dropdown menu for Assay Information select Probe Mix Triplex and complete as follows and click “Apply”:
At this point, if sample names were not added at the start of the run, fill in the sample name by selecting the wells, typing in sample ID and click “Apply”.
If wells need to be deselected: Go to the Plate Editor tab, select wells to exclude and click “Clear Selected Well”.
On the 2D Amplitude tab, make sure that all wells are highlighted and select the Threshold Cluster Mode Graph Tool
and draw 2D thresholds around each cluster as follows
Go through every well to ensure that:
- The droplets are designated properly per well
- There are >10,000 droplets per well
If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.
On the 2D Amplitude tab, select Merged Wells on the left side of the page
Click on the Table Menu Button on the right side of the Well Data table and select Export to CSV to export data.
Save the QuantaSoft Analysis
BCoV/PMMoV post-processing analysis
BCoV/PMMoV post-processing analysis
For BCoV and PMMoV analysis:
Open QuantaSoft™ Analysis Pro Software
Open the .qlp file associated with the run
On the Plate Editor tab, select all the wells then in the dropdown menu for Assay Information select Simplex/Duplex and complete as follows and click Apply:
.On the 1D Amplitude tab, make sure that all wells are highlighted and select the Threshold Multiple Wells button to automatically set the threshold between the positive and negative populations
BCoV threshold = 5000
PMMoV threshold = 1700
Go through every well to ensure that:
- The threshold accurately differentiates positive and negative droplets
- There are >10,000 droplets per well
If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.
On the 1D Amplitude tab, select Merged Wells on the left side of the page
Click on the Table Menu Button on the right side of the Well Data table and select Export to CSV to export data. Save the CSV file.
Save the QuantSoft Analysis
Dimensional Analysis and Quality Control
Dimensional Analysis and Quality Control
For dimensional analysis to express the results of each assay in terms of gene copies/dry weight solids:
Begin with the concentration provided by the QuantaSoft software and reported in the CSV, as expressed in gc/uL of reaction.
Note
This concentration is expressed in terms of the total volume of the merged wells, rather than the volume of the template added. Determination of gc/g relies on the ratio of of template in the reaction and mass in the volume eluted from extraction, so when beginning with this value expressed in terms of gc/uL the number of wells merged is not considered in the dimensional analysis.
To determine the concentration in cp/g dry weight:
Note
When using the assays described in the three protocols that make up this process as written:
- B = 20 µL total reaction volume
- A = 5 µL volume template in reaction
- C = 60 µL elution volume from each well in extraction kit
- Z = 0.0225 g wet weight of solids added to each extraction well (300 µl of a suspension of 75 mg wet weight solids / ml of solution)
- Percent solids varies for each sample on each day (typical range 0.05 - 0.3, i.e. 5-30%)
Please note that there are many different ways to conceptualize the dimensional analysis. Please be sure to double check your own calculations to make sure they match with the measurement units from your instrument. For example, our instrument reports results as copies per the entire volume of the rxn, but some instruments may report a different concentration.
Links to two companion protocols to this protocol can be found in the description of this protocol.