Quantification of the SARS-CoV-2 using electronegative membrane filtration and dPCR

Composite primary influent samples are collected over 24 hours with several samples taken each hour.

100mL sub-samples are collected in 2 x 50 mL sterile conical tubes. The samples are stored at 4°C until further processing.

Thaw an aliquot of BCoV^working () -80On ice (see section Preparation of Bovilis Coronavirus).

Add 62µL of BCoV^working to each 50mL wastewater sample and invert to mix.

Adjust the pH to 3.5 using , mixing the sample between titrations.

Setup the filtration apparatus by attaching tubing to the vacuum port of the vacuum flask and to the vacuum pump. Place a microfunnel on top of the vacuum flask stopper.

Use a 25 mL serological pipette to transfer 25mL of wastewater sample into the microfunnel apparatus and turn on the vacuum pump. Once the entire sample has passed through the filter, remove the upper section of the microfunnel; the filter should remain on the lower portion of the microfunnel.

Submerge two forceps in 70% ethanol and sterilize using a lit flame. While leaving the vacuum on to hold the filter in place, use both forceps to gently roll the filter tightly enough to fit inside a 2 mL tube. Place the rolled filter into a Glass Bead tube, a consumable included in the .

Add 500µL of 1x to the Glass Bead tube and vortex to mix.

Warm PM1 from the at 55°C for 5-10 minutes before use. Add 6µL and 600µL PM1 to the Glass Bead tube.

Also add these reagents to an empty Glass Bead with 500µL of to serve as the extraction control.

Place the Glass Bead tubes in balanced positions in the FastPrep-24 Instrument and run 4 cycles of 20 seconds each at 4.5 m/s. Ensure that the spoke plate has been rotated so that the tube lids are held down.

Equipment

Value	Label
MP Biomedicals™ FastPrep -24™ Classic Instrument	NAME
Benchtop homogenizer	TYPE
Fisher Scientific	BRAND
12079310	SKU

Centrifuge the Glass Bead tubes for 1 minute at 16000x g,0h 0m 0s.

Place the spin columns and elution tubes in their appropriate locations on the Qiagen QIAcube rotor adapter. Transfer the supernatant from the Glass Bead tubes into the center tube of the rotor adapter.

Place the QIAcube rotor adapters in the QIAcube centrifuge and follow the instructions on the QIAcube control tablet to set up the shaker rack, reagents, and tips. When setting up the reagents, shake to mix the PM5 buffer. Set the elution volume to 100µL. Start the extraction run.

Equipment

Value	Label
QIAcube Connect	NAME
Automated nucleic acid extraction	TYPE
Qiagen	BRAND
9002864	SKU

When the extraction is completed, cap the elution tubes and begin the dPCR steps or store at -80°C if the dPCR run will occur in the following days.

Discard the used pipette tips and wipe the waste drawer and QIAcube workspace with 70% ethanol. After each run, remove the plastic tube holder and the reagent tray before running 2 cycles of UV decontamination.

Thaw GT-Molecular controls and assay solutions on ice. If necessary, also thaw the extracted RNA on ice. Once thawed, vortex to mix.

Dilute 1µL extracted RNA with 99µL RNase-free water for PMMoV analysis.

Prepare master mixes for PMMoV and N1-N2-BCoV assays. Allow for one extra sample. Vortex to mix.

A	B
Qiagen 4x One-Step Viral RT-PCR Master Mix	10
Qiagen 100x Multiplex Reverse Transcription Mix	0.4
GT Molecular N1-N2-BCoV Assay Solution	2.0
RNase/DNase free water	7.6

N1-N2-BCoV Master Mix

A	B
Qiagen 4x One-Step Viral RT-PCR Master Mix	10
Qiagen 100x Multiplex Reverse Transcription Mix	0.4
GT Molecular PMMoV Assay Solution	2.0
RNase/DNase free water	7.6

PMMoV Master Mix

Pipette 20µL of the appropriate master mix (N1-N2-BCoV or PMMoV) into the wells of a PCR strip tube.

Add 20µL of extracted RNA sample or positive control to the PCR strip tube following the planned layout. Use the 1:100 diluted samples for the wells being used for the PMMoV assay. After transferring, pipette gently to mix. Keep the PCR strips on ice while loading.

For the non-template control: pipette 20µL of molecular grade water into a PCR tube in place of adding extracted RNA.

Place a Qiagen QIAcuity 26k 24-well Nanoplate onto the Nanoplate protection tray. If the tray is not used, dust can collect on the bottom side of the plate and interfere with the imaging step. Occasionally wipe the tray with 70% ethanol to clean dust.

Using a multichannel pipette, transfer 39µL of solution from the PCR strips to their respective location on the Nanoplate. Be careful to not transfer air bubbles during this step.

Carefully seal the Nanoplate with a Nanoplate seal and the roller provided with the QIAcuity Instrument.

Place the sealed plate in the plate drawer inside the QIAcuity instrument.

Equipment

Value	Label
QIAcuity One, 5-plex	NAME
dPCR	TYPE
Qiagen	BRAND
911021	SKU

Setup the plate by selecting "New Plate". Name the plate and choose the plate type "Nanoplate 26k 24-well".

In the dPCR Parameters section under the Priming tab, select the Qiagen Standard Priming Profile.

Under the Cycling tab create the cycling conditions shown below. These are the conditions recommended by the GT Molecular Wastewater Surveillance Guide.

In the Imaging tab create the conditions shown below. These are GT Molecular recommended.

Navigate to "Reaction mixes". Create reaction mixes named "N1-N2-BCoV Triplex Assay" and "PMMoV Assay" that contain the following details.

Navigate to "Samples and controls". Add samples names that are being quantified on this run. Extraction controls should be added as samples. Under the "Controls" tab create both a "N1-N2-BCoV Positive Control" and a "PMMoV Positive Control". Under the Non Template Controls" tab create a "N1-N2-BCoV dPCRNeg" and a "PMMoV dPCRNeg".

Navigate to "Plate Layout". Assign reaction mixes, samples and controls to their wells. Save plate and exit the setup.

On the QIAcuity tablet, select the plate and run the reaction.

When the QIAcuity run is complete, ensure the image transfer is marked as complete in the QIAcuity Software Suite before inspecting the data.

Open the plate results by selecting "Analysis". Select all the wells and targets before selecting "Show results".

In the 1D Scatterplot tab verify that the automatic threshold is accurately placed between the negative and positive partitions. If needed, adjust the threshold placement to the accurate position.

Use the "Export to CSV" button in the List tab to export the data.

is lyophilized when received. Reconstitute the virus in 5mL pre-chilled molecular grade water and swirl to mix.

Aliquot 100µL stock in sterile 1.5 mL tubes and label each tube BCoV^ND (non-diluted). Store BCoV^ND at-80°C

Dilute 60µL BCoV^ND with 540µLpre-chilled molecular grade water and pipette to mix. Label this tube BCoV^INT (intermediate).

Dilute 500µL BCoV^INT with 49.5mL pre-chilled molecular grade water and invert to mix. Aliquot 1mL of BCoV^working into sterile 1.5 mL tubes and store aliquots at -80°C.

To quantify the BCoV spike, extract an aliquot of BCoV^working in triplicate.

Add 100µL of BCoV^working working into three 1.5 mL tubes.

To each tube add 6µL 2-mercaptoethanol and 600µL PM1 from the Qiagen AllPrep PowerViral DNA/RNA Kit and invert to mix.

Microcentrifuge the tubes at 13000x g,0h 0m 0s for 1 minute.

Add the supernatant to the center column of a rotor adapter and continue the extraction in the Qiagen QIAcube.

Quantify the extracted BCoV^working RNA by analyzing the extraction triplicates using the same dPCR steps beginning in the Detection and Quantification section .

To calculate BCoV^working concentration, use the average measured concentration in copies/microL of the 3 replicates analyzed by dPCR.