TITLE: High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR
AUTHORS: Aaron Topol (Verily Life Sciences), marlene.wolfe  , Brad White (Verily Life Sciences), Krista  Wigginton, Alexandria B B Boehm

[DESCRIPTION]
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. 

[STEPS]
SECTION: Preparation (both assays)
1. 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.

  

SECTION: Preparation (both assays)
2. 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

SECTION: Preparation (both assays)
3. 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.

SECTION: Preparation (both assays)
4. Keep extracted RNA samples on ice or in a cold block from the freezer at all times.

 

SECTION: ddPCR master mix preparation (SARS-CoV-2 targets)
5. Prepare SARS-CoV-2 Target Master Mix

 

SECTION: ddPCR master mix preparation (SARS-CoV-2 targets)
5.1. Prepare a working stock of fresh nuclease free water by pouring from a 50 mL Ambion Nuclease Free water into a 5mL eppendorf tube.
SECTION: ddPCR master mix preparation (SARS-CoV-2 targets)
5.2. Store Master Mix components on ice as much as possible during the preparation process. 

SECTION: ddPCR master mix preparation (SARS-CoV-2 targets)
5.3. 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.

  
SECTION: ddPCR master mix preparation (SARS-CoV-2 targets)
5.4. In a 2mL eppendorf tube, prepare the master mix according to the table for SARS-CoV-2 quantification. Store prepared master mix on ice.

 


 

SECTION: ddPCR master mix preparation (BCoV and PMMoV)
6. Prepare Master Mix for the BCoV/PMMoV assay

 

SECTION: ddPCR master mix preparation (BCoV and PMMoV)
6.1. 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).

SECTION: ddPCR master mix preparation (BCoV and PMMoV)
6.2. In a 2mL eppendorf tube, prepare the master mix according to the table for PMMoV/BCoV quantification. Store prepared master mix on ice.



 

 

SECTION: ddPCR plate set up (BCoV and PMMoV)
7. Dilute the RNA 1:100 for BCoV/PMMoV RT-ddPCR


 

 

SECTION: ddPCR plate set up (BCoV and PMMoV)
7.1. 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”.

SECTION: ddPCR plate set up (BCoV and PMMoV)
7.2. Manually pipette 2 µL of RNA from the stock RNA plate into each corresponding well in the dilution plate.

SECTION: ddPCR plate set up (BCoV and PMMoV)
7.3. Seal the plate and briefly vortex the plate using a bench top plate vortexer.
SECTION: ddPCR plate set up (BCoV and PMMoV)
7.4. Briefly spin down the plate in the Axygen Plate Centrifuge.

SECTION: ddPCR plate set up (BCoV and PMMoV)
8. Store plate on iceuntil ready to add sample to ddPCR plate.

SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
9. For each assay, remove a white cold block from the freezer and place a new Bio-Rad ddPCR 96-well Plate in it.
 


SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
10. Using a new reagent reservoir, manually pipette 16.5 µL of the appropriate Master Mix to each well of each ddPCR Plate.
SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
11. 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 µLof 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)
 

The plate layout should be: 
  

 

SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
12. 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.
 

SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
13. Align a PCR Plate Heat Seal Foil on top of the ddPCR plate with the red line facing up.
SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
14. Seal the plate by pressing the green “seal” button.
SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
15. Briefly vortex the plate using a bench top plate vortexer.

SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
16. Briefly spin down the plate in the bench top Axygen Plate Centrifuge.
SECTION: Transfer Master Mix and Samples to ddPCR plate (both assays)
17. Store the ddPCR plate on a white cold block or on ice until droplet generation.

SECTION: ddPCR droplet generation (both assays)
18. Generate Droplets:
SECTION: ddPCR droplet generation (both assays)
18.1. On the AutoDG droplet generator: select “configure plate” and then press the blue arrow in the upper left corner to highlight all columns.

SECTION: ddPCR droplet generation (both assays)
18.2. 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.

SECTION: ddPCR droplet generation (both assays)
18.3. Place the sealed, vortexed, and centrifuged ddPCR plate in the Sample Plate position on the AutoDG deck.

SECTION: ddPCR droplet generation (both assays)
18.4. Remove the AutoDG cooling block from the freezer and place it in the Droplet Plate slot on the AutoDG deck.

SECTION: ddPCR droplet generation (both assays)
18.5. For the Droplet Plate: label a new Bio-Rad ddPCR™ 96-Well Plate  and place it in the black cooling block.

SECTION: ddPCR droplet generation (both assays)
18.6. Click “Generate Droplets” to begin droplet generation. Droplet generation will take approximately 45 min.
 



SECTION: ddPCR droplet generation (both assays)
18.7. 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.

SECTION: ddPCR droplet generation (both assays)
18.8. Align a PCR Plate Heat Seal Foil on top of the droplet plate with the red line facing up.
  

SECTION: ddPCR droplet generation (both assays)
18.9. Seal the plate by pressing the green “seal” button.

 

SECTION: Thermocycling (SARS-CoV-2)
19. Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for SARS-CoV-2 ddPCR. 

   

SECTION: Thermocycling (SARS-CoV-2)
19.1. 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.

SECTION: Sample storage
20. Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for BCOV/PMMoV ddPCR.
  


SECTION: Sample storage
21. 
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. 

SECTION: Read Droplets (both assays)
22. 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.
SECTION: Read Droplets (both assays)
23. 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. 
SECTION: Read Droplets (both assays)
24. Name the file with a name clearly describing which plate you are reading.
SECTION: Read Droplets (both assays)
25. 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.
SECTION: Read Droplets (both assays)
26. 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”.
SECTION: SARS-CoV-2 Post-Processing Analysis
27. SARS-CoV-2 triplex assay analysis
SECTION: SARS-CoV-2 Post-Processing Analysis
27.1. Open QuantaSoft™ Analysis Pro Software
SECTION: SARS-CoV-2 Post-Processing Analysis
27.2. Open the .qlp file associated with the run

SECTION: SARS-CoV-2 Post-Processing Analysis
27.3. 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”:

  

SECTION: SARS-CoV-2 Post-Processing Analysis
27.4. 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”.
SECTION: SARS-CoV-2 Post-Processing Analysis
27.5. If wells need to be deselected: Go to the Plate Editor tab, select wells to exclude and click “Clear Selected Well”.
SECTION: SARS-CoV-2 Post-Processing Analysis
27.6. 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

  

SECTION: SARS-CoV-2 Post-Processing Analysis
27.7. Go through every well to ensure that:
The droplets are designated properly per well
There are >10,000 droplets per well 
SECTION: SARS-CoV-2 Post-Processing Analysis
27.8. If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.
SECTION: SARS-CoV-2 Post-Processing Analysis
27.9. On the 2D Amplitude tab, select Merged Wells on the left side of the page

SECTION: SARS-CoV-2 Post-Processing Analysis
27.10. Click on the Table Menu Button on the right side of the Well Data table and select Export to CSV to export data. 

SECTION: SARS-CoV-2 Post-Processing Analysis
27.11. Save the QuantaSoft Analysis
SECTION: BCoV/PMMoV post-processing analysis
28. For BCoV and PMMoV analysis:
SECTION: BCoV/PMMoV post-processing analysis
28.1. Open QuantaSoft™ Analysis Pro Software
SECTION: BCoV/PMMoV post-processing analysis
28.2. Open the .qlp file associated with the run

SECTION: BCoV/PMMoV post-processing analysis
28.3. 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:
  

SECTION: BCoV/PMMoV post-processing analysis
28.4. .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
SECTION: BCoV/PMMoV post-processing analysis
28.5. Go through every well to ensure that:
- The threshold accurately differentiates positive and negative droplets 
- There are >10,000 droplets per well 
SECTION: BCoV/PMMoV post-processing analysis
28.6. If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.

SECTION: BCoV/PMMoV post-processing analysis
28.7. On the 1D Amplitude tab, select Merged Wells on the left side of the page
SECTION: BCoV/PMMoV post-processing analysis
28.8. 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.

SECTION: BCoV/PMMoV post-processing analysis
28.9. Save the QuantSoft Analysis
SECTION: Dimensional Analysis and Quality Control
29. For dimensional analysis to express the results of each assay in terms of gene copies/dry weight solids:
SECTION: Dimensional Analysis and Quality Control
29.1. Begin with the concentration provided by the QuantaSoft software and reported in the CSV, as expressed in gc/uL of reaction.
 

SECTION: Dimensional Analysis and Quality Control
29.2. To determine the concentration in cp/g dry weight: