TITLE: One-Step RT-ddPCR for Detection of SARS-CoV-2, Bovine Coronavirus, and PMMoV RNA in RNA Derived from Wastewater or Primary Settled Solids v2 
AUTHORS: Stephanie Loeb, Katy Graham, David Catoe, Marlene Wolfe, Alexandria Boehm, Krista Wigginton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is an updated version of the original protocols.io protocol for these procedures from this group, with changes in the cycling conditions for the BCoV/PMMoV assay and the concentration of the PMMoV probe.</div><div class = "text-block"><span style = "font-weight:bold;">Goal</span><span>: Quantify concentration of N1, N2, BCoV, and PMMoV in RNA extractions using RT-ddPCR. </span></div></div>

[STEPS]
?. [Plan Analysis - Plate Setup]
The goal of this procedure is to quantify concentration of N1, N2, BCoV, and PMMoV in extractions using RT-ddPCR. Two duplexed assays will be run for each sample and are described in this protocol: 1) SARS-CoV-2 N1/N2, and 2) BCoV and PMMoV. Samples from a single plant should be run together on a plate, with the follow types of samples and controls run on each plate: NTC, Standards/Positive Controls, and unknowns.
NTC: No template control; molecular grade water is added to the reaction in place of the template. Double the number of wells run for each sample should be run as NTCs.
Standards/Positive Controls: While a standard curve is not constructed for ddPCR, a positive control confirms that the assay is working and can be used to gauge interplate variability. A positive control is used for each assay, in this case:SARS-CoV-2 N1/N2: The standard is pre-extracted clinical patient RNA diluted in molecular grade water.BCoV: The standard is a direct extraction of 200 µL of reconstituted BCoV vaccine used for spiking diluted in molecular grade water to 106 cp/mL.PMMoV: The standard is a synthetic DNA or RNA ultramer. The following sequence was used as an RNA PMMoV positive control template (Haramoto et al. 2013): tttcaaatgagagtggtttgaccttaacgtttgagaggcctaccgaagcaaatgtcgcacttgcattgcaaccgacaa.
?. [Plan Analysis - Plate Setup]
In order to maximize the likelihood of detecting and quantifying SARS-CoV-2 targets, run at least 2 wells for each sample at 2 dilutions (undiluted and 1:10). Once several samples have been run for a given plant, it may be possible to reduce the number of wells or dilutions depending on the desired limits of detection and quantification and experience with inhibition and anticipated SARS-CoV-2 concentrations at the plant.
Running 3 wells for each sample at 2 dilutions will require:6 wells per sample   of eluent for each sample
Run on each plate (for 3 wells for each sample at 2 dilutions):NTCs (x6)Clinical RNA (x3)6 wells from the same plant:            Undiluted (x3)            Diluted 1:10 (x3)
?. [Plan Analysis - Plate Setup]
The following primer and probe squences, reaction chemistry, and cycling conditions are used for the N1/N2 assays.  ABCD1  <b style="mso-bidi-font-weight:
  normal">Chemistry                 2  Stock Solution    Initial Conc.    Final Conc.    µL / rxn  3  Supermix    4 X    1 X    5.5&nbsp;   4  RT    10 X    1 X    2.2  5  DTT    300mM    15mM    1.1  6  N1/N2 Duplex
  Assay Mix    4 X    1 X    5.5  7  Template              5.5  8  Molecular-grade H20              2.2  9  <b style="mso-bidi-font-weight:
  normal">Total    <b style="mso-bidi-font-weight:
  normal">     <b style="mso-bidi-font-weight:
  normal">     <b style="mso-bidi-font-weight:
  normal">22    ABCD1  <i style="mso-bidi-font-style:
  normal">Cycling Phase    <i style="mso-bidi-font-style:
  normal">Temp °C    <i style="mso-bidi-font-style:
  normal">Time    <i style="mso-bidi-font-style:
  normal"># Cycles  2  RT    50    60 min    1  3  Enzyme Activation    95    10 min    1  4  Denature    94    30 sec       40  5  Anneal/Extension    <b style="mso-bidi-font-weight:
  normal">55    1 min  6Enzyme deactivation  98    10 min    1  7Droplet Stabilization  4    30 min    1  8  Hold    4    ∞    1
Primers and ProbesN1Forward Primer N1-F: 5’-GAC CCC AAA ATC AGC GAA AT-3’’Reverse Primer N1-R: 5’-TCT GGT TAC TGC CAG TTG AAT CTG-3’Probe N1-P-FAM: ACC CCG CAT TAC GTT TGG TGG ACC (5' FAM/ZEN/3' IBFQ)N2Forward Primer N2-F: 5’-TTA CAA ACA TTG GCC GCA AA-3’Reverse Primer N2-R: 5’-GCG CGA CAT TCC GAA GAA-3’Probe N2-P-HEX: ACA ATT TGC CCC CAG CGC TTC AG (5' HEX/ZEN/3' IBFQ)
ABCD1  <b style="mso-bidi-font-weight:
  normal">Chemistry                 2  Stock Solution    Initial Conc.    Final Conc.    µL / rxn  3  Supermix    4 X    1 X    5.5&nbsp;   4  RT    10 X    1 X    2.2  5  DTT    300mM    15mM    1.1  6  N1/N2 Duplex
  Assay Mix    4 X    1 X    5.5  7  Template              5.5  8  Molecular-grade H20              2.2  9  <b style="mso-bidi-font-weight:
  normal">Total    <b style="mso-bidi-font-weight:
  normal">     <b style="mso-bidi-font-weight:
  normal">     <b style="mso-bidi-font-weight:
  normal">22
ABCD1  <i style="mso-bidi-font-style:
  normal">Cycling Phase    <i style="mso-bidi-font-style:
  normal">Temp °C    <i style="mso-bidi-font-style:
  normal">Time    <i style="mso-bidi-font-style:
  normal"># Cycles  2  RT    50    60 min    1  3  Enzyme Activation    95    10 min    1  4  Denature    94    30 sec       40  5  Anneal/Extension    <b style="mso-bidi-font-weight:
  normal">55    1 min  6Enzyme deactivation  98    10 min    1  7Droplet Stabilization  4    30 min    1  8  Hold    4    ∞    1
?. [Plan Analysis - Plate Setup]
In order to maximize the likelihood of detecting and quantifying BCOV and PMMoV targets, running a 1:10 and 1:1000 dilution, respectively, is recommended. Both dilutions may be run with the duplex assays, or single assays can be run with each dilution. Run 2 wells for each sample at each dilution. Please note that once several samples have been run for a given plant, there may be a need to adjust the number of wells or dilutions.
Running 2 wells for each sample at 2 dilutions will require:4 wells per sample per plate  of eluent for each sample
Run on each plate (for 2 wells for each sample at 2 dilutions):NTCs (x4)BCoV/PMMoV Standard (x2)Samples:            Diluted 1:10 (x2)            Diluted 1:1000 (x2)
?. [Plan Analysis - Plate Setup]
The following primer and probe squences, reaction chemistry, and cycling conditions are used for the BCoV/PMMoV assays.  ABCD1               2  Stock Solution    Initial Conc.    Final Conc.    µL / rxn  3  Supermix    4 X    1 X    5.5&nbsp;   4  RT    10 X    1 X    2.2  5  DTT    300mM    15mM    1.1  6  N1/N2 Duplex
  Assay Mix    4 X    1 X    5.5  7  Template              5.5  8  Molecular-grade H20              2.2  9Total22  ABCD1Cycling PhaseTemp °CTime# Cycles2  RT    50    60 min    1  3  Enzyme Activation    95    10 min    1  4  Denature    94    30 sec       40  5  Anneal/Extension  56  1 min  6  Enzyme
  deactivation    98    10 min    1  7  Droplet
  Stabilization    4    30 min    1  8  Hold    4    ∞    1
Primers and ProbesBCoVForward Primer BCoV-F: 5’-CTGGAAGTTGGTGGAGTT-3’’Reverse Primer BCoV-R: 5’-ATTATCGGCCTAACATACATC-3’Probe BCoV-P-FAM: CCTTCATATCTATACACATCAAGTTGTT (5' FAM/ZEN/3' IBFQ)PMMoVForward Primer PMMoV-F: 5’-GAGTGGTTTGACCTTAACGTTTGA-3’Reverse Primer PMMoV-R: 5’-TTGTCGGTTGCAATGCAAGT-3’Probe PMMoV-P-HEX: CCTACCGAAGCAAATG(5' HEX/ZEN/3' IBFQ)(NOTE: performance of the ddPCR assay for PMMoV may be improved with use of an MGB rather than IBFQ quencher)
ABCD1               2  Stock Solution    Initial Conc.    Final Conc.    µL / rxn  3  Supermix    4 X    1 X    5.5&nbsp;   4  RT    10 X    1 X    2.2  5  DTT    300mM    15mM    1.1  6  N1/N2 Duplex
  Assay Mix    4 X    1 X    5.5  7  Template              5.5  8  Molecular-grade H20              2.2  9Total22
ABCD1Cycling PhaseTemp °CTime# Cycles2  RT    50    60 min    1  3  Enzyme Activation    95    10 min    1  4  Denature    94    30 sec       40  5  Anneal/Extension  56  1 min  6  Enzyme
  deactivation    98    10 min    1  7  Droplet
  Stabilization    4    30 min    1  8  Hold    4    ∞    1
?. [Retrieve Samples and Prepare Workspace]
Fill in ddPCR worksheet with samples and controls.
?. [Retrieve Samples and Prepare Workspace]
Sterilize workspace and equipment: UV pipettes, tube racks and molecular grade water, bleach bench and ice buckets (let sit ~ 10 min), turn on PCR hood UV light, wipe bench and ice buckets with 70% EtOH.
?. [Retrieve Samples and Prepare Workspace]
Turn on plate sealer (times out after 2 hours, make sure block is not in machine) and put ice in small bucket for PCR reagents and large bucket for samples.
?. [Retrieve Samples and Prepare Workspace]
Set up tubes for dilutions according to worksheet (tubes will need  of molecular grade water).
27 µl
?. [Retrieve Samples and Prepare Workspace]
Retrieve samples for the day from the -80C freezer. Use a fresh aliquot; each aliquot should only undergo one freeze/thaw cycle before RT-ddPCR.
?. [Reagents and mastermix preparation]
Gather reagents and thaw all components on ice for at least : One Step RT-ddPCR Advanced Kit for Probes (stored in )SupermixReverse transcriptase (RT)300 mM dithiothreitol (DTT) solutionDuplex assay mix of primers and probes (pre-prepared and stored at )
-20 °C
4 °C
?. [Reagents and mastermix preparation]
Mix thoroughly by vortexing each tube to ensure homogeneity (concentration gradient can form when stored below freezing) and briefly spin down to collect contents at the bottom of the tube.
?. [Reagents and mastermix preparation]
In the PCR hood, prepare reaction master mix according to ddPCR worksheet, listed for N1/N2 in step 3  or BCoV-PMMoV from  .
?. [Reagents and mastermix preparation]
Dispense equal aliquots of  into each reaction well.
16.5 µl
Due to the autoDG process, all columns in use must be completely filled. If any wells in a column are empty, fill with 22 µL ddPCR Buffer Control for Probes (catalog. #1863052) diluted 1:1 with water.
?. [Template preparation and dilution]
Add 3 µL of sample to each of the 1:10 dilutions tubes containing 27 µL MGW. Vortex and spin down the 1:10 dilution.
To keep RNA cold through-out, plates/samples can be prepared on a cold block/on ice
?. [Template preparation and dilution]
FOr BCoV/PMMoV targets, continue to dilute to 1:100 and 1:1000. Vortex and spin down.
?. [Template preparation and dilution]
Load samples into the plate (5.5 µL template per well) according to ddPCR worksheet from part 1.
?. [Droplet Generation]
Prep the autoDG: Ensure the trash container is emptyEnsure there is a ready cooling block that has been stored at -20°C for at least 2 hours
?. [Droplet Generation]
Use the plate sealer to seal the sample plate with a heat seal cover.
Place the red strip at top, line up with bottom there should be no overlap at the top or bottom, but the seal will slide up when the drawer shuts.
?. [Droplet Generation]
Vortex plate for 30 seconds, then centrifuge plate at
Centrifuge: 1000 33, 1 min
?. [Droplet Generation]
Gather  autoDG materials and load them:DG32 cartridges (required: one green strip per column, check directionality, will turn green when seated)AutoDG pipet tips in racks (required: two columns of pipets or sample column)Check the level of the AutoDG oil for probesRemove the cooling black accessory from the freezer and place in the droplet plate locationEmpty droplet plate (new ddPCR plate into which the generated droplets will be dispensed) loaded into the cooling block
?. [Droplet Generation]
Load the sample plate into the automated droplet generator in the sample plate position, making sure it is fully seated. The indicator light should turn green.
?. [Droplet Generation]
Touch the configure sample plate button on the AutoDG interface, select the columns in which the samples are located, and touch “OK”. Enter a plate name, and run the droplet generator.
?. [Droplet Generation]
Within 5 minutes of it finishing droplet generation, remove the droplet containing plate from the cold block and seal in the plate sealer. Transfer to the thermocycler.
?. [Thermocycling]
Run the appropriate protocol (N1/N2 from step 3  or BCoV-PMMoV from step 5  ). Confirm the protocol matches the worksheet.
The protocols are identical, except N1/N2 has an annealing temperature of 55°C, while BCoV-PMMoV has an annealing temperature of 60°C.
?. [Thermocycling]
The plate can be transferred to the droplet reader immediately or left at  on the thermal cycler overnight and run on the droplet reader in the morning. While the thermocycler is running, prepare the droplet reader program as described below.
4 °C
Leaving the plate on the droplet reader overnight may improve droplet separation and plate reading.
?. [Droplet Reader]
Open QuantaSoft software (NOT QuantaSoft Analysis Pro).
?. [Droplet Reader]
Load template from previous plate of the same assay - using an old template ensures that all parameters (dye, absorbance etc.) are correct. Save as with a new plate name.
?. [Droplet Reader]
Change sample names (NTCs and controls should be the same)
?. [Droplet Reader]
Double click on A1 - this highlights the name and brings you to the screen where you can change the names (and other parameters)
?. [Droplet Reader]
Change name and click apply or hit enter. Click on the next cell to change (use mouse to click on the next cell). Fill in the entire column, then cut and paste for triplicates (you have to use control C, control V, right clicking with the mouse won’t work)
?. [Droplet Reader]
Make sure the Supermix is set to One-Step RT-ddPCR kit for probes.
This is the only parameter that cannot be changed after the run - if it is not set, the plate reader will fail and the samples will be wasted.
?. [Droplet Reader]
Click ok (next to apply) to get back to the save screen. Save as and double check the name
?. [Droplet Reader]
Open the droplet leader and remove the old plate if it is still inside
?. [Droplet Reader]
Prime the droplet reader before first plate of the day – “Prime” is in the Instrument Routines panel in the top right
?. [Droplet Reader]
Transfer the sample plate from the thermocycler to the droplet reader - it is inserted by sandwiching the plate inside the holder and holding the clips downward.
?. [Droplet Reader]
Hit run. The droplet reader takes 90 seconds per well, or almost 2 ½ hours for a full plate.
If the run won’t start and you don’t see Instrument Routines panel in top right, close program ensure QX200 is plugged in and reopen)
?. [Exporting Data]
When the droplet reader has finished, close QuantaSoft (the program used to run the droplet reader) and open QuantaSoft Analysis Pro.
?. [Exporting Data]
Got to Browse - under QuantaLife>Data you will see folders containing the plate names. Select the folder for your plate and double click the QuantaSoft Plate file. This will open the file in QuantaSoft Analysis Pro.
?. [Exporting Data]
In QuantaSoft Analysis Pro go to File>Save As and save to a USB key as a .ddpcr file. Data can now be managed and exported for downstream analyses.
?. [Appendices]
AppendixPreparing ddPCR assay mixPrimer and probes will arrive  either lyophilized, or as 100 µM suspended in TE. Resuspend all primers and probe to 100 µM in molecular grade TE, and combine in the appropriate ratio, dilute with molecular grade water and store at -20°C. [NB: Currently, SARS CoV-2 primers and FAM labeled-probe are only available from IDT as 100 µM pre-suspended in TE.]When plates are being frequently run, assay mix can be stored at 4°C for up to 2 weeks. Recommended to defrost on ice, or overnight at 4°C when possible.
?. [Appendices]
Prepare the bench top area for use – wipe surface and equipment with 10% bleach, allow to dry for at least 10 min, and wipe with 70% ethanol.
?. [Appendices]
Add molecular grade TE buffer to any lyophilized primers and probes according to the moles designated on the tube to make 100 µM solutions. (e.g. add 348 µL of TE to 34.8 nmol of primer).
?. [Appendices]
SARS CoV-2 4X assay mix (): In a 2 mL LoBind epi-tubes combine  N1-F,  N1-R,      N2-F,  N2-R,  N1-P-FAM, and  N2-P-HEX. Add  molecular grade water and store at -20°C until ready for use.
1 mL
36 µl
36 µl
36 µl
36 µl
10 µl
10 µl
836 µl
?. [Appendices]
BCoV-PMMoV 4X assay mix (): In a 2 mL LoBind epi-tubes combine  BCoV-F,  BCoV-R,  PMMoV-F,  PMMoV-R, BCoV-P-FAM, and  PMMoV-P-HEX. Add molecular grade water to increase solution to () and store at until ready for use.
1 mL
36 µl
36 µl
36 µl
36 µl
10 µl
16 µl
1 mL
-20 °C