TITLE: Quantification of the SARS-CoV-2 using Nanotrap Particles®, the QIAcuity™ Digital PCR System, and GT-Digital SARS-CoV-2 Wastewater Surveillance Assay
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 Nanotrap particles, RNA extraction using 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 as well as the Ceres Nano Manual Nanotrap Wastewater Protocol using Qiagen AllPrep PowerViral DNA/RNA Kit protocol (Ceres Nano).

[STEPS]
SECTION: Sample Collection
1. Composite primary influent samples are collected (grab samples, flow composite, or time composite)
SECTION: Sample Collection
2. 50 mL sub-samples are collected in 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). Pipette up and down to mix the aliquot and briefly centrifuge.

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. Using a serological pipette transfer 10 mL of the wastewater sample into a 15 mL conical tube. Repeat this for a duplicate.

SECTION: Concentration of Viral Fraction
7. Add 100 µL of Nanotrap Enhancement Reagent 2 to each 10 mL wastewater sample and vortex for a few seconds.

SECTION: Concentration of Viral Fraction
8. Add 150 µL of Nanotrap Magnetic Virus Particles and invert 2-3 times to mix. 

SECTION: Concentration of Viral Fraction
9. Incubate the samples for 10 minutes at room temperature.

SECTION: Concentration of Viral Fraction
10. Place the sample tubes in a 15 mL magnetic rack. Wait 1 minute. Remove the supernatant using a serological pipette. The magnetic particles form a pellet on the sides of the tube so it is essential to hold the tip of the pipette in the center of the tube while removing the supernatant. Transfer the supernatant to a waste container.

SECTION: Nucleic Acid Extraction
14. 
 

SECTION: Nucleic Acid Extraction
15. Warm PM1 from the    at 55 °C for 5-10 minutes before use. 

SECTION: Nucleic Acid Extraction
18. Incubate the samples including the extraction control for 10 minutes at room temperature. 



SECTION: Nucleic Acid Extraction
19. Place the 1.5 mL sample tubes on the magnetic rack and wait 1 minute before transferring the supernatant into the center tube of the rotor adapter.



SECTION: Nucleic Acid Extraction
20. 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
21. 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
22. 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
23. 
 

 

SECTION: Detection and Quantification of SARS-CoV-2
24. 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
25. Dilute 1 µL extracted RNA with 99 µL RNase-free water for PMMoV analysis.

SECTION: Detection and Quantification of SARS-CoV-2
26. Prepare master mixes for PMMoV and N1-N2-BCoV assays. Allow for one extra sample. Vortex to mix. Briefly centrifuge the tubes to collect the master mix at the bottom of the tube.
 


 
  
  

SECTION: Detection and Quantification of SARS-CoV-2
27. 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
28. 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
29. 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
30. 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
31. 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
32. Carefully seal the Nanoplate with a Nanoplate seal and the roller provided with the QIAcuity Instrument.
SECTION: Detection and Quantification of SARS-CoV-2
33. Place the sealed plate in the plate drawer inside the QIAcuity instrument. 

 

SECTION: Detection and Quantification of SARS-CoV-2
34. 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
35. In the dPCR Parameters section under the Priming tab, select the Qiagen Standard Priming Profile.
SECTION: Detection and Quantification of SARS-CoV-2
36.   
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
37. In the Imaging tab create the conditions shown below. These settings are different from the GT Molecular recommended settings, but provides less frequent saturation errors.

   

SECTION: Detection and Quantification of SARS-CoV-2
38. 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
39. 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
40. 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
41. On the QIAcuity tablet, select the plate and run the reaction.
SECTION: Analysis and Interpretations
42. 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
43. Open the plate results by selecting "Analysis". Select all the wells and targets before selecting "Show results".
SECTION: Analysis and Interpretations
44. 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
45. Use the "Export to CSV" button in the List tab to export the data.
SECTION: Index: Preparation of Bovilis Coronavirus
46. 
 

SECTION: Index: Preparation of Bovilis Coronavirus
47.   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
48. 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
49. Dilute 60 µL BCoVND with 540 µLpre-chilled molecular grade water and pipette to mix. Briefly centrifuge. Label this tube BCoVINT (intermediate). 

SECTION: Index: Preparation of Bovilis Coronavirus
50. 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
51. To quantify the BCoV spike, extract the RNA of an aliquot of BCoVworking in triplicate.  
SECTION: Index: Preparation of Bovilis Coronavirus
51.1. Add 100 µL of BCoVworking working into three 1.5 mL tubes. 

SECTION: Index: Preparation of Bovilis Coronavirus
51.2. 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
51.3. Microcentrifuge the tubes at 13000 x g for 1 minute. 

SECTION: Index: Preparation of Bovilis Coronavirus
51.4. 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
52. 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
53. To calculate BCoVworking concentration, use the average measured concentration in copies/microL of the 3 replicates analyzed by dPCR.
  

SECTION: Concentration of Viral Fraction
11. Add 1 mL molecular grade water to each 15 mL tube and remove from magnetic rack. Invert the sample to collect the Nanotrap Magnetic Particles in the water.

SECTION: Concentration of Viral Fraction
12. Transfer the sample using a pipet to a 1.5 mL tube. Place the 1.5 mL tubes on a 1.5 mL magnetic rack for 1 minute to allow the magnetic particles to separate from the supernatant. Discard the supernatant.
SECTION: Concentration of Viral Fraction
13. Add 200 µL   to each sample and vortex to mix. If extraction is delayed, store samples up to 3 days at 4 °C .

SECTION: Nucleic Acid Extraction
16. Add  6 µL   and 600 µL  PM1 to each 1.5 mL tube containing the concentrated sample and Nanotrap Magnetic Virus Particles. Vortex to mix.



SECTION: Nucleic Acid Extraction
17.  Prepare an extraction control by adding  6 µL  , 600 µL  PM1, and 200 µL RNA Shield to an empty tube. Also add 100 µL Nanotrap Enhancement Reagent 2 and 150 µL Nanotrap Magnetic Virus Particles. 

SECTION: Index: Imaging Errors
54. The QIAcuity instrument may show an error when viewing results that a "channel has reached saturation". To resolve this error, reduce the imaging gain and exposure duration and re-image the plate.