TITLE: Quantification of the SARS-CoV-2 using electronegative membrane filtration and dPCR
AUTHORS: Solana  Narum, Thibault  Stalder, Erik  Coats, Eva Top

[DESCRIPTION]
This protocol was developed in an effort to serve as a timely and efficient method for the surveillance of the SARS-CoV-2 in primary influent wastewater samples. This process describes viral concentration via electronegative adsorption with pH adjustment, RNA extraction using the MP Biomedicals FastPrep-24 and the Qiagen AllPrep PowerViral DNA/RNA kits with the Qiacube Connect, and quantification of the N1 and N2 genes in SARS-CoV-2 using the GT Digital SARS-CoV-2 Wastewater Surveillance for QIAcuity.

To compile the entire process from beginning to the end some sections were taken from the AllPrep PowerViral DNA/RNA Kit (Qiagen) and the GT Digital SARSCoV- 2 Wastewater Surveillance for QIAcuity v1.0 (GT Molecular) handbooks.

[STEPS]
SECTION: Sample Collection
1. Composite primary influent samples are collected over 24 hours with several samples taken each hour. 
SECTION: Sample Collection
2. 100 mL sub-samples are collected in 2 x 50 mL sterile conical tubes. The samples are stored at 4 °C until further processing.

SECTION: Concentration of Viral Fraction
3. 
 

SECTION: Concentration of Viral Fraction
4. Thaw an aliquot of BCoVworking ( ) on ice (see section Preparation of Bovilis Coronavirus).

SECTION: Concentration of Viral Fraction
5. Add 62 µL of BCoVworking to each 50 mL wastewater sample and invert to mix.

SECTION: Concentration of Viral Fraction
6. Adjust the pH to 3.5 using  , mixing the sample between titrations.

SECTION: Concentration of Viral Fraction
7. 
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. 
SECTION: Concentration of Viral Fraction
8. Use a 25 mL serological pipette to transfer 25 mL 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.

SECTION: Concentration of Viral Fraction
9. 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  .

SECTION: Concentration of Viral Fraction
10. Add 500 µL of 1x  to the Glass Bead tube and vortex to mix. 

SECTION: Nucleic Acid Extraction
11. 
 

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

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

SECTION: Nucleic Acid Extraction
13. 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.


 


SECTION: Nucleic Acid Extraction
14. Centrifuge the Glass Bead tubes for 1 minute at 16000 x g.



SECTION: Nucleic Acid Extraction
15. 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. 
SECTION: Nucleic Acid Extraction
16. 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 100 µL. Start the extraction run.

 


SECTION: Nucleic Acid Extraction
17. 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.

SECTION: Nucleic Acid Extraction
18. 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.
SECTION: Detection and Quantification of SARS-CoV-2
19. 
 

 

SECTION: Detection and Quantification of SARS-CoV-2
20. Thaw GT-Molecular controls and assay solutions on ice. If necessary, also thaw the extracted RNA on ice. Once thawed, vortex to mix.
SECTION: Detection and Quantification of SARS-CoV-2
21. Dilute 1 µL extracted RNA with 99 µL RNase-free water for PMMoV analysis.

SECTION: Detection and Quantification of SARS-CoV-2
22. Prepare master mixes for PMMoV and N1-N2-BCoV assays. Allow for one extra sample. Vortex to mix.
 


 
  
  

SECTION: Detection and Quantification of SARS-CoV-2
23. Pipette 20 µL of the appropriate master mix (N1-N2-BCoV or PMMoV) into the wells of a PCR strip tube.

SECTION: Detection and Quantification of SARS-CoV-2
24. Add 20 µL 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.

SECTION: Detection and Quantification of SARS-CoV-2
25. For the non-template control: pipette 20 µL of molecular grade water into a PCR tube in place of adding extracted RNA.

SECTION: Detection and Quantification of SARS-CoV-2
26. 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.
SECTION: Detection and Quantification of SARS-CoV-2
27. Using a multichannel pipette, transfer 39 µL of solution from the PCR strips to their respective location on the Nanoplate. Be careful to not transfer air bubbles during this step.
 


SECTION: Detection and Quantification of SARS-CoV-2
28. Carefully seal the Nanoplate with a Nanoplate seal and the roller provided with the QIAcuity Instrument.
SECTION: Detection and Quantification of SARS-CoV-2
29. Place the sealed plate in the plate drawer inside the QIAcuity instrument. 

 

SECTION: Detection and Quantification of SARS-CoV-2
30. Setup the plate by selecting "New Plate". Name the plate and choose the plate type "Nanoplate 26k 24-well".
SECTION: Detection and Quantification of SARS-CoV-2
31. In the dPCR Parameters section under the Priming tab, select the Qiagen Standard Priming Profile.
SECTION: Detection and Quantification of SARS-CoV-2
32.   
Under the Cycling tab create the cycling conditions shown below. These are the conditions recommended by the GT Molecular Wastewater Surveillance Guide.

   

SECTION: Detection and Quantification of SARS-CoV-2
33. In the Imaging tab create the conditions shown below. These are GT Molecular recommended.  

  

SECTION: Detection and Quantification of SARS-CoV-2
34. Navigate to "Reaction mixes". Create reaction mixes named "N1-N2-BCoV Triplex Assay" and "PMMoV Assay" that contain the following details.

  
  

SECTION: Detection and Quantification of SARS-CoV-2
35. 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".
SECTION: Detection and Quantification of SARS-CoV-2
36. Navigate to "Plate Layout". Assign reaction mixes, samples and controls to their wells. Save plate and exit the setup.
SECTION: Detection and Quantification of SARS-CoV-2
37. On the QIAcuity tablet, select the plate and run the reaction.
SECTION: Analysis and Interpretations
38. When the QIAcuity run is complete, ensure the image transfer is marked as complete in the QIAcuity Software Suite before inspecting the data.
SECTION: Analysis and Interpretations
39. Open the plate results by selecting "Analysis". Select all the wells and targets before selecting "Show results".
SECTION: Analysis and Interpretations
40. 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.
SECTION: Analysis and Interpretations
41. Use the "Export to CSV" button in the List tab to export the data.
SECTION: Index: Preparation of Bovilis Coronavirus
42. 
 

SECTION: Index: Preparation of Bovilis Coronavirus
43.   is lyophilized when received. Reconstitute the virus in 5 mL pre-chilled molecular grade water and swirl to mix. 

SECTION: Index: Preparation of Bovilis Coronavirus
44. Aliquot 100 µL stock in sterile 1.5 mL tubes and label each tube BCoVND (non-diluted). Store BCoVND at-80 °C

SECTION: Index: Preparation of Bovilis Coronavirus
45. Dilute 60 µL BCoVND with 540 µLpre-chilled molecular grade water and pipette to mix. Label this tube BCoVINT (intermediate).

SECTION: Index: Preparation of Bovilis Coronavirus
46. Dilute 500 µL BCoVINT with 49.5 mL pre-chilled molecular grade water and invert to mix. Aliquot 1 mL of BCoVworking into sterile 1.5 mL tubes and store aliquots at -80 °C.

SECTION: Index: Preparation of Bovilis Coronavirus
47. To quantify the BCoV spike, extract an aliquot of BCoVworking in triplicate.  
SECTION: Index: Preparation of Bovilis Coronavirus
47.1. Add 100 µL of BCoVworking working into three 1.5 mL tubes. 

SECTION: Index: Preparation of Bovilis Coronavirus
47.2. To each tube add 6 µL 2-mercaptoethanol and 600 µL PM1 from the Qiagen AllPrep PowerViral DNA/RNA Kit and invert to mix.

SECTION: Index: Preparation of Bovilis Coronavirus
47.3. Microcentrifuge the tubes at 13000 x g for 1 minute. 

SECTION: Index: Preparation of Bovilis Coronavirus
47.4. Add the supernatant to the center column of a rotor adapter and continue the extraction in the Qiagen QIAcube.
SECTION: Index: Preparation of Bovilis Coronavirus
48. Quantify the extracted BCoVworking RNA by analyzing the extraction triplicates using the same dPCR steps beginning in the Detection and Quantification section .
SECTION: Index: Preparation of Bovilis Coronavirus
49. To calculate BCoVworking concentration, use the average measured concentration in copies/microL of the 3 replicates analyzed by dPCR.