Mar 28, 2022
Version 2
Quantification of the SARS-CoV-2 using electronegative membrane filtration and dPCR V.2
- Solana Narum1,
- Thibault Stalder1,
- Erik Coats1,
- Eva Top1
- 1University of Idaho
Protocol Citation: Solana Narum, Thibault Stalder, Erik Coats, Eva Top 2022. Quantification of the SARS-CoV-2 using electronegative membrane filtration and dPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61xwrvqe/v2Version created by Solana Narum
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We currently use this protocol, but are also investigating other methods that may provide more reliable data.
Created: March 28, 2022
Last Modified: March 28, 2022
Protocol Integer ID: 60005
Keywords: SARS-CoV-2, Wastewater, Wastewater Based Epidemiology
Funders Acknowledgement:
National Institute of General Medical Sciences of the National Institutes of Health
Grant ID: P20GM104420
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Abstract
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.
Materials
Equipment:
Equipment
MP Biomedicals™ FastPrep -24™ Classic Instrument
NAME
Benchtop homogenizer
TYPE
Fisher Scientific
BRAND
12079310
SKU
LINK
Equipment
QIAcube Connect
NAME
Automated nucleic acid extraction
TYPE
Qiagen
BRAND
9002864
SKU
LINK
Equipment
QIAcuity One, 5-plex
NAME
dPCR
TYPE
Qiagen
BRAND
911021
SKU
LINK
Reagents:
Bovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
Hydrochloric acidSigma Aldrich
Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
2-MercaptoethanolSigma Aldrich
Qiagen AllPrep PowerViral DNA/RNA Extraction KitQiagenCatalog #28000-50
Qiagen QIAcuity One-Step Viral RT-PCR KitQiagenCatalog #1123145
GT-Digital SARS-CoV-2 Wastewater Surveillance Assay for QIAcuityGT Molecular
Consumables:
50 mL conical tubes, sterile, nuclease-free
Pall Microfunnel Filter Funnel with GN-6 Membrane, 100 mL, 0.45 m, sterile (Fischer Scientific, Catalog #: NC0553687)
Qiagen Sample Tubes (Qiagen, Catalog #: 990381)
Qiagen Rotor Adapters (Qiagen, Catalog #: 990394)
Qiagen QIAcuity Filter Tips, 1000 microL (Qiagen, Catalog #: 990352)
PCR tubes, RNase free, low-binding
Qiagen QIAcuity Nanoplate 26k 24-well (Qiagen, Catalog #: 250002)
In addition to the materials listed above, this protocol requires access to typical microbiology equipment including pipettes, sterile, nuclease-free, filter barrier pipette tips, microcentrifuge, a Class II biosafety cabinet, a PCR cabinet, a vacuum pump, vacuum flask and stopper, vortexes, and standard PPE.
Protocol materials
Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
In Materials and 2 steps
2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)
Materials
Qiagen AllPrep PowerViral DNA/RNA Extraction KitQiagenCatalog #28000-50
In Materials and 2 steps
Bovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
In Materials and 2 steps
Hydrochloric acidMerck MilliporeSigma (Sigma-Aldrich)
Materials, Step 6
Qiagen QIAcuity One-Step Viral RT-PCR KitQiagenCatalog #1123145
Materials, Step 22
GT-Digital SARS-CoV-2 Wastewater Surveillance Assay for QIAcuityGT Molecular
Materials, Step 22
2-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
Step 12
Safety warnings
Prior to processing wastewater samples for the detection of the SARS-CoV-2, work with your local biosafety committee to establish biosafety protocols, and review the CDC Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19) available at https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html#environmental
Sample Collection
Sample Collection
1d
1d
Composite primary influent samples are collected over 24 hours with several samples taken each hour.
100 mL sub-samples are collected in 2 x 50 mL sterile conical tubes. The samples are stored at 4 °C until further processing.
Concentration of Viral Fraction
Concentration of Viral Fraction
1h
1h
Note
These steps should be performed in a Class II Biosafety Cabinet, which has been cleaned with RNase Away and water.
Thaw an aliquot of BCoVworking (Bovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445 ) On ice (see section Preparation of Bovilis Coronavirus).
Add 62 µL of BCoVworking to each 50 mL wastewater sample and invert to mix.
Adjust the pH to 3.5 using Hydrochloric acidSigma Aldrich , 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 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.
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 Qiagen AllPrep PowerViral DNA/RNA Extraction KitQiagenCatalog #28000-50 .
Add 500 µL of 1x Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706 to the Glass Bead tube and vortex to mix.
Nucleic Acid Extraction
Nucleic Acid Extraction
2h
2h
Safety information
Proper aseptic technique should be used when working with RNA. Always wear powder-free latex, vinyl, or nitrile gloves while handling reagents, tubes, and RNA samples to prevent RNase contamination from the surface of the skin or from the environment. Change gloves frequently and keep tubes closed. During the procedure work quickly and keep everything on cold blocks when possible, to avoid degradation of RNA by endogenous or residual RNAses. Clean working surfaces and pipettes with RNase away.
The first step should be performed in the Class II Biosafety Cabinet decontaminated with UV light and RNase away. After the lysis solutions have been applied in step 9, the additional steps can take place outside of the Biosafety Cabinet.
Warm PM1 from the Qiagen AllPrep PowerViral DNA/RNA Extraction KitQiagenCatalog #28000-50 at 55 °C for 5-10 minutes before use. Add 6 µL 2-mercaptoethanolSigma AldrichCatalog #M6250 and 600 µL PM1 to the Glass Bead tube.
Also add these reagents to an empty Glass Bead with 500 µL of Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706 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
MP Biomedicals™ FastPrep -24™ Classic Instrument
NAME
Benchtop homogenizer
TYPE
Fisher Scientific
BRAND
12079310
SKU
LINK
Centrifuge the Glass Bead tubes for 1 minute at 16000 x g .
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 100 µL . Start the extraction run.
Equipment
QIAcube Connect
NAME
Automated nucleic acid extraction
TYPE
Qiagen
BRAND
9002864
SKU
LINK
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.
Detection and Quantification of SARS-CoV-2
Detection and Quantification of SARS-CoV-2
4h
4h
Note
Technical notes:
The dPCR instrument is sensitive to microbubbles. To prevent errors in quantification and imaging, ensure that pipettes only reach the first stop when preparing this reaction.
Safety information
The preparation of the dPCR plate should be done in a PCR workstation previously decontaminated by UV light and has been cleaned with RNase away and water.
Thaw GT-Molecular controls and assay solutions on ice. If necessary, also thaw the extracted RNA on ice. Once thawed, vortex to mix.
Dilute 1 µL extracted RNA with 99 µL RNase-free water for PMMoV analysis.
Prepare master mixes for PMMoV and N1-N2-BCoV assays. Allow for one extra sample. Vortex to mix.
| N1-N2-BCoV Master Mix Components | Volume/sample (microL) | |
| 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
| PMMoV Master Mix Components | Volume/sample (microL) | |
| 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
GT-Digital SARS-CoV-2 Wastewater Surveillance Assay for QIAcuityGT Molecular
Qiagen QIAcuity One-Step Viral RT-PCR KitQiagenCatalog #1123145
Pipette 20 µL of the appropriate master mix (N1-N2-BCoV or PMMoV) into the wells of a PCR strip tube.
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.
For the non-template control: pipette 20 µL 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 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.
Note
Do not push the pipette beyond the first stop 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
QIAcuity One, 5-plex
NAME
dPCR
TYPE
Qiagen
BRAND
911021
SKU
LINK
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.
Analysis and Interpretations
Analysis and Interpretations
15m
15m
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.
Index: Preparation of Bovilis Coronavirus
Index: Preparation of Bovilis Coronavirus
1h
1h
Note
The Bovilis Coronavirus (BCoV) is used as internal process control and is added to each sample prior to filtration and extraction.
It is necessary to quantify BCoV for each new batch of BCoVworking is made from a new tube of BCoVND.
BCoV should not be vortexed at any point in the preparation or spiking of samples.
Bovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445 is lyophilized when received. Reconstitute the virus in 5 mL pre-chilled molecular grade water and swirl to mix.
Aliquot 100 µL stock in sterile 1.5 mL tubes and label each tube BCoVND (non-diluted). Store BCoVND at-80 °C
Dilute 60 µL BCoVND with 540 µL pre-chilled molecular grade water and pipette to mix. Label this tube BCoVINT (intermediate).
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 .
To quantify the BCoV spike, extract an aliquot of BCoVworking in triplicate.
Add 100 µL of BCoVworking working into three 1.5 mL tubes.
To each tube add 6 µL 2-mercaptoethanol and 600 µL PM1 from the Qiagen AllPrep PowerViral DNA/RNA Kit and invert to mix.
Microcentrifuge the tubes at 13000 x g 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 BCoVworking RNA by analyzing the extraction triplicates using the same dPCR steps beginning in the Detection and Quantification section .
To calculate BCoVworking concentration, use the average measured concentration in copies/microL of the 3 replicates analyzed by dPCR.