Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials
2.1.1. Wastewater Collection
- Disposable polypropylene centrifuge tubes, 50 mL (Thermo Fish Scientific, Waltham, MA, USA; Cat. no.: 05-539-13)
2.1.2. Wastewater Incubation
- Kimwipes delicate task wipers 1-ply (Kimberly-Clark, Irving, TX, USA; Cat. no.: 06-666)
- 75% ethanol
- 50 mL tube racks
2.1.3. Wastewater Filtration
- Filter system, 150 mL, 0.22 µm PES membrane (Corning Inc., Corning, NY, USA; Cat. no.: 431153)
2.1.4. Virus Concentration
- Amicon Ultra-15 centrifugal filters (Merck Millipore, Carrigtwohill, CO, USA; Cat. no.: UFC901096D)
2.1.5. Nucleic Acid Extraction
- QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany; Cat. no.: 52906)
- Rotor adapters (Qiagen; Cat. no.: 990394)
- Filter-tips 1000 µL (Qiagen; Cat. no.: 990352)
2.1.6. Molecular Detection (RT-qPCR)
- Quantinova Probe RT-PCR Kit (Qiagen; Cat. no.: 990352)
- Strip tubes and caps, 0.1 mL (Qiagen; Cat. no.: 981103)
- SARS-CoV-2 CDC RUO primers and probes (Integrated DNA Technologies, Coralville, IA, USA; Cat. no.: 10006713)
- 2019-nCoV_N_positive control (Integrated DNA Technologies; Cat. no.: 10006625)
2.2. Equipment
2.2.1. Wastewater Collection
- Portable sampler unit (Teledyne ISCO, Lincoln, NE, USA; Cat. no.: 6837000069 and 699003588)
2.2.2. Wastewater Incubation
- Bead bath dry bath (Lab Armor LLC, Irving, TX, USA; Cat. no.: SL-74309-720)
2.2.3. Wastewater Filtration
- Class II biosafety cabinet (Labconco, Kansas City, MO, USA; Cat. no.: 36208)
2.2.4. Virus Concentration
- Allegra X-12R tabletop centrifuge (Beckman Coulter, Brea, CA, USA; Cat. no.: 392472)
2.2.5. Nucleic Acid Extraction
- Digital vortex mixer (Thermo Fish Scientific, Waltham, MA, USA; Cat. no.: 02-215-418)
- QIAcube Connect automated extraction unit (Qiagen; Cat. no.: 9002864)
2.2.6. Molecular Detection (RT-qPCR)
- PCR Workstation (CBS Scientific, East Lyme, CT, USA; Cat. no.: P-030-202)
- Rotor-Gene Q 2-Plex system apparatus (Qiagen; Cat. no.: 9001620)
3. Procedure
3.1. Wastewater Collection. Time for Completion: 24:00 h
- Collect wastewater at hourly intervals manually or through programming a portable sampler unit over a twenty-four-hour period. If wastewater is collected from a location which is only occupied during specific hours (e.g., schools or businesses), then collect during that time only.
- Thoroughly mix the composited wastewater, then transfer 40–45 mL into two 50 mL centrifuge tubes and tightly cap the tubes.
- Transport samples to the laboratory and decontaminate the exterior of the tubes using 75% ethanol, or equivalent.
3.2. Wastewater Incubation. Time for Completion: 01:00 h
- Invert the sample tubes several times to suspend settled solids then place the tubes in the bead bath, which is pre-heated to 60 °C.
-
- CRITICAL STEP Incubation should be performed to not only decrease the risks of processing human wastewater but also to homogenize the sample and keep the solids in suspension while processing. Greater recovery (p < 0.05; data not shown) of SARS-CoV-2 was observed after incubation was performed.
-
- Incubate the wastewater for thirty minutes, invert tubes to re-suspend settled solids, then return the samples to the bead bath for an additional thirty minutes.
- After incubation for the full sixty minutes is completed, remove the samples from the bead bath.
-
- PAUSE STEP Allow wastewater to cool by storing samples at refrigeration temperature (4 °C) prior to performing filtration (Step 3.3). Water above ambient temperature may impact the PES membrane of the filter unit. Samples may be stored up to 72 h at 4 °C before continuing the processing; however, concentration should be completed to reach the frozen storage stage without exceeding this timeframe.OPTIONAL STEP Remove aliquots (>1 mL) of incubated wastewater for detection of normalization organisms (Scheme 1, Steps 5* and 6*). Detection of additional organisms can be performed using the method continued below; however, considerations then need to be made to account for variations in recovery of the organisms due to their morphological differences.
-
3.3. Wastewater Filtration. Time for Completion: 00:30 min
- Transfer 40–45 mL of wastewater from one tube into a 0.22 µm Corning filter unit.
- Connect the filter to a vacuum system and allow the wastewater to pass through the membrane until a minimum of 30 mL can be collected as filtrate and the large particles have been removed.
-
- CRITICAL STEP Filtration of the wastewater is necessary to remove particles which may cause inefficient concentration; increased turbidity and volume of viral concentrates (3.4) were observed when filtration was omitted. Additionally, nucleic acid extraction (3.5) efficiency may decrease or inhibition of molecular detection (3.6) may occur.
-
3.4. Virus Concentration. Time for Completion: 01:00 h
- Collect 30 mL of filtrate and divide evenly between two Amicon 10 kDa centrifugal filters.
- Place the Amicon filters into the centrifuge and spin for 45 min at 3000× g at ambient temperature.
- Collect the viral concentrate from the two filters, and pool the concentrate for each sample in a microcentrifuge tube.
-
- PAUSE STEP The protocol may be paused here by storing viral concentrates at −20 °C until nucleic acid extraction is performed. If samples are frozen, they must be thawed to room temperature prior to nucleic acid extraction.
-
3.5. Nucleic Acid Extraction. Time for Completion: 02:00 h
- Perform extractions in duplicate for each sample using QIAamp Viral RNA Mini Kit and the QIAcube automated extraction instrument per the manufacturer’s instructions.OPTIONAL STEP Nucleic acid extractions may be performed manually in lieu of using the automated system by following the manufacturer’s instructions.OPTIONAL STEP RNA and DNA of additional target organisms can be extracted simultaneously using Qiagen’s RNeasy PowerMicrobiome Kit (Cat. no.: 26000-50). Recovery efficacy of other virus, bacteria, or phage should be monitored separately from SARS-CoV-2 due to morphological differences. Larger organisms should be processed directly after incubation (3.2) or eluted from the filter (3.3).
- (a)
- Add a filter column from the QIAamp kit to each rotor adapter in position 1 and 1.5 mL collection tube from the Rotor Adapter set in position 3 then place the Rotor Adapters into the centrifuge of the QIAcube.
- (b)
- Prepare the Buffer AVL-carrier RNA solution (560 µL Buffer and 5.6 µL RNA per sample) and add 560 µL to two microcentrifuge tubes for each sample.
- (c)
- Pipette to mix the virus concentrate and add 140 µL to two tubes containing the Buffer AVL-carrier RNA solution.
- (d)
- Pulse-vortex the samples for 15 s and incubate at room temperature for 10-min.
- (e)
- While samples are incubating, transfer 700 µL from the microcentrifuge tubes to CB tubes.
- (f)
- Place the CB tubes in the QIAcube and follow the on-screen instructions for the QIAamp Viral RNA Mini Kit, adjusting the elution volume to 50 µL and keeping all other options at the manufacturer’s recommended settings.
- Remove the Rotor Adapters from the QIAcube centrifuge when the run is complete and place in a biosafety cabinet.
- Discard the filter column, close the collection tube and transfer to a microcentrifuge tube rack. Discard the waste in the bottom of the Rotor Adapter into a flammable waste container for chemical disposal.
-
- PAUSE STEP The protocol may be paused here by storing nucleic acids at −20 °C until detection is performed. If samples are frozen, prior to detection they must be thawed on ice before adding RNA template to the RT-qPCR reaction. If long-term storage of RNA is required, transfer to −80 °C.
-
3.6. Molecular Detection (RT-qPCR). Time for Completion: 02:00 h
- Allow reagents, and nucleic acids if necessary, to thaw.
- Prepare the reaction mix using 10 µL of Master Mix, 0.2 µL of Reverse Transcriptase Mix, 1 µL of each primer and probe, and water to a total volume of 17 µL per reaction tube. Primers and probes should be at 8000 and 4000 nM stock concentrations, respectively to achieve the concentrations shown in Table 1 for the reaction mix.
- Transfer 17 µL of the reaction mix to each tube, then add template RNA, negative and positive controls.
- Cap the strip tubes and place in the Rotor-Gene Q.
- Program the instrument to run at 45 °C for 10 min, 95 °C for 5 min, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Set to acquiring during the 60 °C period for both green (FAM) and yellow (HEX) channels.
3.7. Data Interpretation and Analyses
- After the RT-qPCR run is complete, perform the in-system analysis by setting values for the standard curve or importing an existing curve, then setting the threshold.
- Export the data and calculate the average viral copies/L using the N1 and N2 target copies/reaction data. Report data with no amplification above the threshold as below the limit of quantification or limit of detection, which for this protocol is 10,000 copies/L.
4. Expected Results
5. Reagents Setup
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target | Sequence (5′-3′) 1 | Conc. (nM) |
---|---|---|
N1 | F: GAC CCC AAA ATC AGC GAA AT | 400 |
R: TCT GGT TAC TGC CAG TTG AAT CTG | 400 | |
P: FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1 | 200 | |
N2 | F: TTA CAA ACA TTG GCC GCA AA | 400 |
R: GCG CGA CAT TCC GAA GAA | 400 | |
P: FAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1 | 200 |
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Anderson-Coughlin, B.L.; Shearer, A.E.H.; Omar, A.N.; Wommack, K.E.; Kniel, K.E. Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration. Methods Protoc. 2021, 4, 32. https://doi.org/10.3390/mps4020032
Anderson-Coughlin BL, Shearer AEH, Omar AN, Wommack KE, Kniel KE. Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration. Methods and Protocols. 2021; 4(2):32. https://doi.org/10.3390/mps4020032
Chicago/Turabian StyleAnderson-Coughlin, Brienna L., Adrienne E. H. Shearer, Alexis N. Omar, K. Eric Wommack, and Kalmia E. Kniel. 2021. "Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration" Methods and Protocols 4, no. 2: 32. https://doi.org/10.3390/mps4020032