TITLE: Concentration, extraction and quantification of SARS-CoV-2 in wastewater via droplet digital PCR
AUTHORS: Jim Huang, August Luc, Sarah Kane, Rose Nash, Susan De Long, Carol Wilusz

[DESCRIPTION]
This protocol describes the method used at Colorado State University to extract and quantify SARS-CoV-2 in wastewater collected from sewers and at wastewater treatment plants.  The method relies on concentration via ultrafiltration, silica column based purification, and quantification via digital droplet reverse transcription PCR.  Endogenous and exogenous controls are described.

[STEPS]
SECTION: Preparation
1. Sample Description and Treatment
This protocol is designed for use with untreated influent from wastewater treatment plants.  Samples should be provided in 50ml conical tubes with ~40ml of influent in each tube.  Optimally, the samples should be flow proportional, collected over a 24 hour time period, and stored at ~4°C for transport to the laboratory for processing.  The protocol is also appropriate for grab samples and for flow/time proportional samples collected from sewers.
 

 

SECTION: Preparation
2. Work Area Set Up
Wipe down Biosafety Cabinet (BSC) with 70% ethanol
Thaw sufficient BCoV spike for the number of samples [(N+2)x 14.5μl] - store on ice
Perform Start Up Procedure on Innovaprep and insert effluent tube into beaker containing 10% bleach 
Ensure sufficient Elution Fluid, Ultrafiltration Tips etc are available to complete the run
 


SECTION: Preparation
3. 
Bovine Coronavirus Spike-In   
  
Lyophilized BCoV (10 dose) is reconstituted in 5 ml PBS, 0.01% Tween 20 and stored in aliquots at -80°C
A working stock at 1:500 dilution in PBS, 0.01% Tween 20 is prepared and stored in 500 μl aliquots at -80°C 
The concentration of the working stock should be in the range of 3000 genome copies per μl

 

SECTION: Clarification and Concentration of Wastewater
4. Sample Pre-Treatment
Addition of BCoV allows virus recovery and inhibition of PCR due to the wastewater matrix to be detected.  Tween-20 is added as recommended by Innovaprep.  Perform this step on ice.

Thaw the BCoV aliquot on ice 
Vortex and centrifuge at 16,000g for 5 minutes 
Transfer the supernatant to a fresh tube.  This is to ensure the viral particles are evenly distributed.
Adjust the volume of each wastewater sample to 40 ml
Add 400 μl of 10% Tween 20
Add 14.3 μl of BCoV spike (~30-50,000 genome copies)
Mix well by inverting
Store remaining BCoV spike on ice until Step 6 is complete
5. Filtration to remove particulates
  
Clarification of the sample is necessary to avoid clogging at the ultrafiltration step.  This protocol uses filtration but centrifugation can also be employed and is cheaper.
Pass each sample through a 0.22μM PES filter (diameter >8 cm)
6. Innovaprep Ultrafiltration
  
  
Insert a fresh ultrafiltration tip into the device
Insert the tip into the sample
Start Run (see note for program details)
If run stops before all liquid is aspirated, Press Return and Start Run again.  This is more likely to happen with turbid samples, or if the tip is faulty.
Remove the empty sample bottle and replace with a 15 ml conical tube
Press Elute
Store the eluate on ice until all samples have been concentrated
Transfer 140 μl of eluate to a fresh tube containing 560 μl of Qiagen AVL buffer and mix well before proceeding to Step 8
The remaining eluate can be stored at -80°C

 



 

 




SECTION: Spike Lysis
7. Spike Lysis
 
Add 5μl Quick Extract DNA Extraction Solution to 10μl BCoV Spike from Step 5
Heat in thermocycler at 65°C for 15 min then 98°C for 2 min, then 4°C forever
Add 85 μl H2O and store on ice for use in ddPCR
Can store at -80°C for reference
 

SECTION: Extraction of RNA
8. QiaAMP Viral RNA Extraction Protocol

 
After 10 min incubation, briefly spin down the AVL/Eluate mix
Add 560 μl 100% ethanol
Vortex for 15 sec
Apply 630 μl to Epoch column in a 2ml collection tube 
Centrifuge 6,000xg for 1 minute.
Decant the filtrate
Load the remaining sample onto the column
Centrifuge 6,000xg for 1 minute
Add 500μl of AW1 and spin 1 minute at 6,000xg for 1 minute
Add 500μl of AW2 and spin at max speed for 3 minutes
Place the column in a fresh 2mL collection tube and spin again for 1 minute at max speed
Place the column in a fresh, labeled 1.5ml tube and add 60μl of RNase free water to each column
Incubate for 1 minute 
Spin at 6,000xg for 3 minutes
Store samples on ice (if using immediately) or at -80°C for long term storage
 

 

SECTION: Preparation for ddPCR
9. SARS-CoV-2 Positive Control
This reagent is used as a positive control for ddPCR detection of SARS-CoV-2.  The variant listed below works well with the primer set described here, other variants may or may not be detected.
 
   
Add 5μl Quick Extract DNA Extraction Solution to 10μl heat-inactivated virus 
Heat in thermocycler at 65°C for 15 min then 98°C for 2 min, then 4°C forever
Add 85 μl H2O 
Dilute to desired concentration, aliquot and store at -80°C
 


SECTION: Preparation for ddPCR
10. Primer / Probe Mixes

See Materials for Sequences.
Primer/probe mixes are made as described below, aliquoted, and stored at -20°C in the dark.  After thawing on ice, aliquots are stored at 4°C.

100 μM Forward Primer    100 μl
100 μM Reverse Primer    100 μl
100 μM Probe                       25 μl
H2O                                    1275 μl
SECTION: One-Step Reverse Transcription Droplet Digital PCR
11. Reaction Set Up

Thaw all required reagents on ice:
> Reverse Transcriptase
> 300mM DTT
> Primer Probe Mixes
> BioRad 1-step RT-ddPCR supermix for probes
> Wastewater RNA 
> Positive controls

SARS-CoV-2 /BCoV Reactions
Create a mastermix sufficient for all reactions adding reagents in the order shown 
Include 1-2 safety reactions to ensure there is enough mastermix i.e. if you have 10 reactions make a mastermix for 11-12 reactions

 

Pipette each template RNA into one well of an 8-tube strip.
Add 13µl  mastermix to each well
Vortex briefly and spin to the bottom of the tube

F+ / PMMoV Reactions
Dilute template RNA 1:100 for F+ and PMMoV reactions - add 3 μl RNA to 273 μl H2O
Create a mastermix sufficient for all reactions adding reagents in the order shown 
Include 1-2 safety reactions to ensure there is enough mastermix i.e. if you have 10 reactions make a mastermix for 11-12 reactions

 

Pipette each diluted template RNA into one well of an 8-tube strip.
Add 13µl  mastermix to each well
Add ddPCR buffer control for probes to any empty wells in the 8 tube strip
Mix and spin to the bottom of the tube

It is important to include both positive and negative controls.  The negative control should contain H2O instead of RNA template.  
SECTION: One-Step Reverse Transcription Droplet Digital PCR
12. Droplet Generation

Place a DG8 cartridge in the holder and click to close
Using a multi-channel pipette, transfer 20 μl of each reaction to the "Sample" row
Dispense 70 μl AutoDG* (droplet generator) oil for probes in the "Oil" row
Seal with a clean gasket, insert into droplet generator and press Start
Using a p200 gently transfer the droplets to a 96-well plate
Repeat until all samples are in the 96-well plate
Turn on the plate sealer.
Place a foil seal on top of the plate.  Seal at 180 °C for 5 sec
 * We find that Auto-DG oil reproducibly gives a higher droplet count than Manual-DG oil, even when using a manual droplet generator.
SECTION: One-Step Reverse Transcription Droplet Digital PCR
13. Thermocycling

Using a BioRad C1000 Touch deep-well device:
50°C for 1 hr  (reverse transcription step)
95°C for 10 min
[94°C for 30 sec; 55°C for 1 min] x 44; 
98°C for 10 min
4°C forever
SECTION: One-Step Reverse Transcription Droplet Digital PCR
14. Droplet Reading

Follow the manufacturer's instructions for using the QX200 reader
Allow 2.5 min per sample plus 10-15 minutes for set up

Open the reader, insert the plate, lock in the metal grid on top and close the device
Open the Quantasoft program
For each well, select ABS (for Absolute Quantification), Ch1 FAM (SARS-CoV-2 or F+), Ch2 HEX (BCoV or PMMoV) and 1-step RT-ddPCR kit for probes.
Click RUN
SECTION: Data Analysis
15. Setting Thresholds in Quantasoft Analysis Pro

Follow the manufacturer's instructions for using the software
On the Droplets tab, select all wells.  Verify that the droplet count is over 10,000 in all wells
 
In the 2D Amplitude view, select all wells containing the SARS/BCoV probes
Position the thresholds (pink +) such that they fall between positive and negative droplets
 
Check on the 1D Amplitude view that the threshold in both channels lies above all the droplets in the no template control.  
Set the thresholds for the F+/PMMoV wells in the same way (but not at the same time because the correct position will be different).

SECTION: Data Analysis
16. Exporting Data from Quantasoft Manager Pro to MS Excel

On 1D or 2D Amplitude view, select all the wells
On the Well Data panel menu select "Export to Excel"
While you can select all the data, the values required for downstream calculations or reporting to the NWSS/CDC are:
> Well
> Sample
> Target
> Copies/20 ul
> Accepted   [# droplets]
> PoissonConfMax
> PoissonConfMin
SECTION: Data Analysis
17. Calculating SARS-CoV-2 Genome Copies per L

The following values are required to convert the copies per 20µl [C] into copies per L:
> Volume of wastewater sample   [W=40ml]
> Total volume of eluate from innovaprep [IP = ranges from 200-900 µl]
> Volume of eluate used in RNA extraction [IE = 140µl] 
> Total volume of RNA eluted after extraction [RT = 60 µl] 
> Volume of RNA used in reaction [RU = 8.2 µl] 

 

Using the protocol described above:
  
 

SECTION: Data Analysis
18. Calculating Genome Copies per L for Endogenous Controls

The same calculation is used as above, but should be multiplied by 100 to take into account that the RNA template for the PMMoV and F+ reaction is diluted 100 fold.
 
 

Using the protocol described above:
  
SECTION: Data Analysis
19. Determining BCoV Recovery

Calculate the copies of BCoV that were added at the start of the protocol:
Use the copies per 20µl in the BCoV positive control reaction to determine how many copies of BCoV were added to each sample at the beginning.

  
  
  

Then calculate the number of copies of BCoV that would have been recovered if you had processed the entire 40ml of wastewater and express as a percentage of the copies that were added.

  
 






SECTION: Data Analysis
20. Quality Controls and Troubleshooting

One possible use of the controls is for normalization.  In our experience with Colorado WWTPs normalization to either exogenous (BCoV) or endogenous controls (PMMoV, F+) did not improve correlations with community infection rate or reduce variability.

The controls can also be used to detect failure of reactions due to inhibitors and to distinguish between inhibition and degradation:
  
Both the reverse transcription step and the PCR step can be inhibited by compounds that carry through the extraction step (either reagents such as phenol or guanidine, or contaminants such as humic acids).  Inhibition of the RT step will generally result in fewer positive droplets (because there will be less cDNA produced) and confounds accurate quantification.  Inhibition of the PCR step can result in reduced fluorescence amplitude i.e. poor separation of the positive and negative droplets, because each cycle will produce fewer copies of the cDNA template).  This may not affect quantification if the separation remains adequate for threshold setting.   Inhibition is not template specific - all targets in a reaction mix will be affected. Low BCoV recovery (<5%) can indicate inhibition of the RT step.  Importantly, inhibition can often be abrogated by using less of the RNA containing the inhibitor.  Therefore the PMMoV/F+ reaction will generally give good results even when inhibitors are present because it uses a 100 fold diluted RNA template.  If BCoV recovery fails QC, it is helpful to rerun the reaction with half or quarter the amount of RNA template (assuming the SARS-N1 levels are high enough to still be detected).

In contrast, degradation of the RNA sample will result in fewer positive droplets for all four targets (i.e. Low BCOV recovery and low PMMoV/F+).  This problem can only be remedied by repeating the extraction to make fresh RNA.

RNA quality cannot be easily evaluated prior to the ddPCR assay because even good quality RNA is generally very low abundance (too low for nanodrop), partially degraded and represents a mixture of nucleic acids from a variety of microbes, viruses and human cells (confounds Tapestation/Bioanalyzer).  Fluorescent measurements (such as Qubit) may be informative but add time and expense.