Jul 01, 2020
Version 2
Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR V.2
- 1Louisiana State University;
- 2CSIRO;
- 3University of Notre Dame
Protocol Citation: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby 2020. Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bhiuj4ew
Manuscript citation:
Ahmed W, Bertsch P, Bivins A, Bibby K, Farkas K, Gathercole A, Haramoto E, Gyawali P, Korajkic A, McMinn BR, Mueller J. Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater. Science of The Total Environment. 2020 Jun 5:139960.
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 use this protocol and it's working
Created: June 15, 2020
Last Modified: July 01, 2020
Protocol Integer ID: 38196
Keywords: wastewater, SARS-CoV-2 RNA, ddPCR,
Disclaimer
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Abstract
The following protocol describes the GERM Lab workflow for processing wastewater for SARS-CoV-2 via pH adjustment, MCE filtration, extraction, and assay by droplet digital PCR (ddPCR). It is synonymous to method A as described in Ahmed et al. 2020 (the referenced manuscript) with modifications to the consumables and PCR format. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.
Graphical abstract of the GERM Lab protocol for concentration SARS-CoV-2 RNA from wastewater and assying by droplet digital PCR.
Guidelines
Please coordinate with your local biosafety committee to execute any wastewater handling and concentration procedures in accordance with biosafety standards for your institute and interim guidance issued by the CDC as referenced in the safety warning.
Materials
MATERIALS
INFORCE 3 Intranasal Bovine VaccineZoetisCatalog #INF-00089
Hydrochloric AcidVWR InternationalCatalog #87003-253
2-MercaptoethanolMP BiomedicalsCatalog #0219470580
STEP MATERIALS
INFORCE 3 Intranasal Bovine VaccineZoetisCatalog #INF-00089
Hydrochloric AcidVWR InternationalCatalog #87003-253
2-MercaptoethanolMP BiomedicalsCatalog #0219470580
RT-ddPCR OligonucleotidesIntegrated DNA TechnologiesCatalog #Varies
2019-nCoV RUO KitIntegrated DNA TechnologiesCatalog #10006713
2019-nCoV_N_Positive ControlIntegrated DNA TechnologiesCatalog #10006625
One-Step RT-ddPCR Advanced Kit for ProbesBioRad SciencesCatalog #186-4021
Droplet Generation Oil for ProbesBioRad SciencesCatalog #1863005
ddPCR™ Droplet Reader OilBioRad SciencesCatalog #1863004
In addition to the reagents listed above and the equipment and consumables listed within, this protocol requires access to typical microbiology equipment including pipettes and tips, microcentrifuge, an autoclave, a Class II biosafety cabinet, central vacuum line, and vortex.
Protocol materials
Droplet Generation Oil for ProbesBio-Rad LaboratoriesCatalog #1863005
Materials, Step 28
2-MercaptoethanolMP BiomedicalsCatalog #0219470580
In Materials, Materials, Step 11
INFORCE 3 Intranasal Bovine VaccineZoetisCatalog #INF-00089
In Materials, Materials, Step 2
2019-nCoV RUO KitIntegrated DNA Technologies, Inc. (IDT)Catalog #10006713
Materials, Step 22.2
One-Step RT-ddPCR Advanced Kit for ProbesBio-Rad LaboratoriesCatalog #186-4021
Materials, Step 23
ddPCR™ Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
Materials, Step 35
2019-nCoV_N_Positive ControlIntegrated DNA Technologies, Inc. (IDT)Catalog #10006625
Materials, Step 22.2
Hydrochloric AcidVWR InternationalCatalog #87003-253
In Materials, Materials, Step 6
RT-ddPCR OligonucleotidesIntegrated DNA Technologies, Inc. (IDT)Catalog #Varies
Materials, Step 22
Safety warnings
Prior to processing wastewater samples for SARS-CoV-2, please review the CDC recommended biosafety guidelines for environmental specimen testing available at https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html#environmental. Work with your local biosafety committee to establish applicable biosafety protocols.
Before start
We are making this protocol available as a courtesy to our colleagues. We make no guarantees or warranties concerning its performance in other laboratories or settings.
Using this procedure at Notre Dame, we have successfully detected and quantified SARS-CoV-2 and pepper mild mottle virus in raw wastewater influent samples via the workflow described herein. Based on results described for murine hepatitis virus by Ahmed et al. 2020 (Method A: mean recovery 27%) adsorption direct extraction with pH adjustment will allow for reasonable recovery of SARS-CoV-2 RNA from wastewater. Please note that it is possible to perform this protocol without acidification (Ahmed et al. 2020 Method B mean recovery 61%), and with MgCl2 amendment (Ahmed et al. 2020 Method C mean recovery 66%). We have chosen to use acidification so that we can compare our results in the current project with our historical sampling results.
This procedure relies on vacuum filtration for up to 4 hours to concentrate a 250 mL wastewater sample. It is advisable to use a central vacuum line for this task. However, it is also possible to filter a 100 mL sample in about 30 minutes.
Sample Collection
Sample Collection
3h
Collect a 1 L sample of primary influent or raw wastewater (grab sample or composite) in a sterile sample collection bottle. Maintain the sample at 4 °C during transport to the lab.
2h
Upon sample receipt at the laboratory spike 1 mL of resuspended INFORCE 3 into the wastewater sample. INFORCE 3 contains bovine respiratory synyctial virus (BRSV) which serves as an RNA process control. BRSV is an enveloped single-stranded RNA virus. Spiking 1 mL of INFORCE 3 into 1 liter of wastewater yields approximately 10,000 BRSV gene copies per mL in the wastewater matrix. Mix thoroughly after spiking INFORCE 3 and allow the sample to incubate at 4 °C for 00:30:00 . Spiking the process control prior to any freezing and thawing allows for the effect of these processes on recovery to be examined. Other enveloped RNA process controls are also in use by other laboratories including murine hepatitis virus and bovine coronavirus.
INFORCE 3 Intranasal Bovine VaccineSigma AldrichCatalog #INF-00089
30m
If sample is to be concentrated immediately temporary storage at 4 °C is sufficient. If sample is to be processed at a future date (more than 2 days later), mix well, aliquot as suggested below, and store at -80 °C until concentration is to be completed. Aliquot 500 mL of the wastewater sample for concentration as described below and store the remaining 500 mL at -80 °C for any future anayses that may be required.
10m
HA Concentration
HA Concentration
10h
Prior to concentration, sterilize the funnel top, funnel stem/membrane support, aluminum clamp, and filtration flask and GN-6 MCE filters by autoclaving. Alternatively, Millipore HAWP MCE filters can also be used in this protocol.
Equipment
HAWP MF-Millipore Membrane Filter, 0.45 µm pore size
NAME
Membrane filter
TYPE
Millipore
BRAND
HAWP04700
SKU
LINK
0.45 um 47 mm
SPECIFICATIONS
Equipment
GN-6 Metricel 0.45 um 47 mm MCE gridded
NAME
membrane filter
TYPE
Pall Corporation
BRAND
63020
SKU
LINK
Equipment
Vacuum Filtration Assembly
NAME
Glassware
TYPE
Sigma-Aldrich
BRAND
Z290408
SKU
LINK
for 47 mm filters with glass support, NS 40/35 joints, 1 L flask (included)
SPECIFICATIONS
1h
Assemble two vacuum filtration assemblies with GN-6 MCE filters for each sample that will be processed (two biological replicates). Handle filters and assemblies using proper aseptic technique. All concentration and processing should be carried out within a Class II Biosafety Cabinet. Attach each filtration flask to a central vacuum line using vacuum tubing. Include a bleach trap to avoid contaminating the vacuum line.
**Note vacuum times to filter the entire volume are up to 8 hours depending on wastewater sample turbidity, so a central vacuum line is strongly recommended.
Filtration columns and bleach traps set up within the BSC.
15m
Using 2N Hydrochloric acid adjust the pH of each 500 mL wastewater sample to be concentrated to ~3 to 4. Add Hydrochloric acid in increments of 100 uL to 10 uL and measure the pH at each step using pH strips and sterile forceps. After acidiciation, gently mix the wastewater sample in a sealed vessel prior to beginning filtration. Please note that the protocol can also be performed with two variations: (1) no acidification netural pH or (2) amendment with MgCl2. Please see Ahmed et al. 2020 for more details on these variations.
Equipment
pH Test Strips 0 - 14
NAME
pH test strips
TYPE
VWR International
BRAND
BDH35309.606
SKU
LINK
Hydrochloric AcidSigma AldrichCatalog #87003-253
15m
Slowly add 250 mL of wastewater sample to a single filtration assembly funnel top in increments of approximately 50 mL . Allow the suspended settles to settle. Add the remaining 250 mL of wastewater sample to a second filtration assembly funnel top in the same manner. After wastewater sample has been added to each filtration assembly, slowly turn on the main vacuum line to begin filtration. Monitor for at least the first minute or two to ensure there is no leakage. Continue the vacuum until the entire 250 mL of each wastewater replicate has passed through the GN-6 MCE filter. Depending on the turbidity of the wastewater, filtration could take up to 8 hours.
Raw influent sample at the beginning of filtration.
Filter with solids caked on after filtration.
8h
After filtration, use a pair of sterilized forceps and aseptic technique to fold the GN-6 MCE filter and slide it into a 2mL PowerBead Tube, Garnet. If the filter is dry, use sterile water to rehydrate the filter prior to folding to prevent breakage.
Equipment
PowerBead Tube, Garnet
NAME
bead beating tube
TYPE
Qiagen
BRAND
13123-50
SKU
LINK
PowerBead Tubes, Garnet 0.70 mm
SPECIFICATIONS
15m
Store filters in PowerBead tubes at -80 °C until extraction.
RNA Extraction
RNA Extraction
3h
RNA Extraction is performed using the Qiagen Allprep PowerViral DNA/RNA Kit with modifications.
Equipment
Allprep PowerViral DNA/RNA Kit
NAME
DNA/RNA extraction kit
TYPE
Qiagen
BRAND
28000-50
SKU
LINK
AllPrep PowerViral DNA/RNA Kit (50)
SPECIFICATIONS
Sample Homogenization: Briefly centrifuge each PowerBead tube ~5000 x g then add 650 µL of Warm Solution PM1 and 6.5 µL Beta-mercaptoethanol (Beta-ME) to each PowerBead tube. Homogenize the samples with 4 cycles for 20 seconds at 4.0 M/s on an MP Bio FastPrep 24. In between each 20 second homogenization cycle, briefly centrifuge the PowerBead tubes ~5000 x g . After the fourth and final homogenization cycle, centrifuge the PowerBead tubes at 13000 x g, 00:01:00 at room temperature. Transfer 450 µL of the resulting supernatant to a clean 2 mL Collection Tube.
2-MercaptoethanolSigma AldrichCatalog #0219470580
Equipment
MP Bio FastPrep 24
NAME
bead beating grinder
TYPE
MP Bio
BRAND
116004500
SKU
LINK
Homogenized filters after bead beating using an MP Bio FastPrep 24.
30m
Add 150 µL of Solution IRS and vortex briefly. Incubate at 4 °C for 00:05:00 .
Centrifuge the 2 mL Collection Tube at 13000 x g, 00:01:00 . Transfer 500 µL of supernatant to a clean 2.2 mL Collection Tube avoiding the pelleted material.
Add 600 µL of Solution PM3 and 600 µL of Solution PM4 to each 2.2 mL Collection Tube and vortex briefly to mix.
Load 625 µL of mixture onto an MB Spin Column and centrifuge at 13000 x g, 00:01:00 . Discare the flow through and repeat until all the supernatant has been filtered through the Spin Column.
Shake to mix Solution PM5 and add 600 µL to the MB Spin Column. Centrifuge at 13000 x g, 00:01:00 .
Discard the flow through and add 600 µL of Solution PM4. Centrifuge at 13000 x g, 00:01:00 . Discard the flow through and centrifuge at 13000 x g, 00:02:00 .
Place the MB Spin Column into a clean 2 mL Collection Tube. Add 80 µL of RNase-free water to the center of the white column membrane and incubate for at least 00:01:00 . Centrifuge at 13000 x g, 00:01:00 and discard the MB Spin Column.
Spin the 2 mL Collection Tube at 13000 x g, 00:02:00 and transfer 60 µL of supernatant to a 1.5 mL Eppendorft LoBind Tube.
Equipment
DNA LoBind Tube 1.5 mL
NAME
Microcentrifuge tube
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
The DNA/RNA is now ready for downstream analaysis. Store at -80 °C until further analysis.
RT-ddPCR Oligos
RT-ddPCR Oligos
The RT-ddPCR is performed using the One-Step RT-ddPCR Advanced Kit for Probes from BioRad. The concise steps below are taken from the package insert with a few modifications. For full details, please reference the BioRad Droplet Digital PCR Applications Guide and the User Manuals for the referenced equipment.
Please note that the protocol excludes details regarding experimental design such as technical replicates, biological replicates, negative extraction controls, no-template controls etc. Users are referred to the digital MIQE Guidelines for recommendations concerning quality assurance and control.
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW. The Digital MIQE Guidelines: M inimum I nformation for Publication of Q uantitative Digital PCR E xperiments. Clinical chemistry. 2013 Jun 1;59(6):892-902.
Primers, probes, and control materials:
Unless otherwise specified, all oligonucleotide materials are purchased from Integrated DNA Technologies (IDT, Corallvile, IA).
RT-ddPCR OligonucleotidesSigma AldrichCatalog #Varies
BRSV primers and probes:
Boxus M, Letellier C, Kerkhofs P. Real Time RT-PCR for the detection and quantitation of bovine respiratory syncytial virus. J Virol Methods. 2005 May;125(2):125-30.
The probe for the BRSV assay specified below is on HEX (Channel 2)
Target region: beta-actin
Amplicon length: 124 bp
| FWD Primer | 5' GCA ATG CTG CAG GAC TAG GTA TAA T 3' | |
| REV Primer | 5' ACA CTG TAA TTG ATG ACC CCA TTC T 3' | |
| Probe | /5HEX/AC CAA GAC T/ZEN/T GTA TGA TGC TGC CAA AGC A/3IABkFQ/ |
Table 1 - BRSV primers and probes for RT-ddPCR
SARS-CoV-2 primers, probes, and control plasmid:
The primer and probes set targeting the N1 and N2 genes are available premixed from IDT. The probes for both the N1 and N2 probes are on FAM (Channel 1).
2019-nCoV RUO KitSigma AldrichCatalog #10006713
The N gene control plasmid is also avialable from IDT.
2019-nCoV_N_Positive ControlSigma AldrichCatalog #10006625
RT-ddPCR
RT-ddPCR
7h
The RT-ddPCR is performed using the One-Step RT-ddPCR Advanced Kit for Probes from BioRad.
The kit referenced below is for 200 reactions and contains:
(1) Supermix: 500 µL x 2
(2) Reverse Transcriptase, RT: 200 µL x 2
(3) Dithiothreitol, DTT solution: 1 mL 300 millimolar (mM) x 2
One-Step RT-ddPCR Advanced Kit for ProbesSigma AldrichCatalog #186-4021
All components on the One-Step RT-ddPCR Advanced Kit for Probes are stable for 12 months when stored at -20 °C . Repeated freezing and thawing of the supermix is not recommended. DTT should be aliquoted to multiple tubes and stored at -20 °C to minimize freezing and thawing.
Thaw all components on ice for 00:30:00 . Mix each component thoroughly by vortexing each tube several times for 00:00:30 to ensure homogeneity because a concentration gradient may form during -20 °C storage. Centrifuge briefly to collect contents at the bottom of each tube.
Prepare samples at the desired concentration before setting up the reaction mix. Store samples at 4 °C while preparing the reaction mix.
Prepare the reaction mix for the number of reactions needed as shown below. The reactions should be setup on ice before droplet generation to prevent a nonspecific reverse transcription reaction from occurring. Assemble all required components except the sample, dispense equal aliquots into each reaction tube or well, and add sample to reach tube or well as the final step.
BRSV reagent volumes per reaction:
Reagent Stock Conc. Reaction Conc. Volume per reaction (uL)
Water n/a n/a 5.59 µL
Supermix 4x 1x 5.25 µL
RT n/a n/a 2.1 µL
DT 300 millimolar (mM) 15 millimolar (mM) 1.05 µL
Probe 100 micromolar (µM) 250 nanomolar (nM) 0.055 µL
Fwd 10 micromolar (µM) 900 nanomolar (nM) 1.98 µL
Rev 10 micromolar (µM) 900 nanomolar (nM) 1.98 µL
WW RNA Variable* Variable* 4 µL
Total Volume per reaction: 22 µL
N1 or N2 reagent volumes per reaction:
Reagent Stock Conc. Reaction Conc. Volume per reaction (uL)
Water n/a n/a 6.45 µL
Supermix 4x 1x 5.25 µL
RT n/a n/a 2.1 µL
DT 300 millimolar (mM) 15 millimolar (mM) 1.05 µL
IDT PreMix Probe @ 250 nM 3.15 µL
Primers @ 1000 nM
WW RNA Variable* Variable* 4 µL
Total Volume per reaction: 22 µL
*Suggested input quantities of total RNA are 100 fg - 100 ng per reaction
Mix thoroughly by vortexing the reaction tubes or, if preparing reactions in a 96-well plate, by pipette mixing 5 times prior to transferring the reaction mix to the droplet generation cartridge.
Once the reaction mixtures are ready, place a DG8 Cartridge into a DG8 Cartridge Holder. Next load 20 µL of each reaction mix into the sample well of a DG8 Cartridge for QX200/QX100 Droplet Generator followed by 70 µL of Droplet Generation Oil for Probes into the oil wells, according to the QX100 or QX200 Droplet Generator Instruction Manual. After loading the sample and droplet generation oil, seal the DG8 cartridge in the DG8 Cartridge Holder using a DG8 Gasket.
Equipment
DG8™ Cartridges for QX200™/QX100™ Droplet Generator
NAME
Droplet Generation Cartridges
TYPE
BioRad
BRAND
1864008
SKU
LINK
Equipment
DG8 Cartridge Holder
NAME
Cartridge Holder
TYPE
BioRad
BRAND
1863051
SKU
LINK
Droplet Generation Oil for ProbesSigma AldrichCatalog #1863005
Equipment
DG8™ Gaskets for QX200™/QX100™ Droplet Generator
NAME
DG8 Gasket
TYPE
BioRad
BRAND
1863009
SKU
LINK
Load the DG8 Cartridge Holder onto the QX200 Droplet Generator and close the lid to begin droplet generation.
Equipment
QX200™ Droplet Generator
NAME
QX200™ Droplet Generator
TYPE
BioRad
BRAND
1864002
SKU
LINK
Once droplet generation is complete, carefully transfer 40 µL of the droplet oil emulsion from the DG8 Cartridge to a 96-well ddPCR plate.
Equipment
ddPCR™ 96-Well Plates
NAME
ddPCR™ 96-Well Plates
TYPE
BioRad
BRAND
12001925
SKU
LINK
Once the droplets from all samples have been transferred to the 96-well ddPCR plate, seal the plate with plate heat seal foil using the PX1 PCR Plate Sealer.
Equipment
PCR Plate Heat Seal, foil, pierceable
NAME
Heat Seal foil
TYPE
BioRad
BRAND
1814040
SKU
LINK
Equipment
PX1 PCR Plate Sealer
NAME
Plate Sealer
TYPE
BioRad
BRAND
1814000
SKU
LINK
After sealing the 96-well plate, incubate the plate at 4 °C for 00:15:00 .
Place the sealed 96-well plate on the C1000 Touch Thermal Cycler and perform thermal cycling as specificed in Table 2 below.
Equipment
C1000 Touch™ Thermal Cycler with 96–Deep Well Reaction Module
NAME
Thermal Cycler
TYPE
BioRad
BRAND
1851197
SKU
LINK
Cycling Step Temperature (C) Time Number of Cycles
Reverse Transcription 50 °C 01:00:00 1
Enzyme Activation 95 °C 00:10:00 1
Denaturation 95 °C 00:00:30 40x
Annealing/Extension 59 °C 00:01:00 above
Enzyme Deactivation 98 °C 00:10:00 1
Hold 4 °C Infinite 1
When thermal cycling is complete, allow the 96-well plate to incubate at 4 °C for 00:20:00 .
Place the 96-well plate in the QX200 Droplet Reader and ensure there is adequate ddPCR Droplet Reader Oil in the reservoir.
Equipment
QX200™ Droplet Reader
NAME
QX200™ Droplet Reader
TYPE
BioRad
BRAND
1864003
SKU
LINK
ddPCR™ Droplet Reader OilSigma AldrichCatalog #1863004
Open QuantaSoft Software to set up a new plate layout according to the experimental design. Refer to the QX100 or QX200 Droplet Reader and QuantaSoft Software Instruction Manual.
Under Setup, double click on a well in the plate layout to open the Well Editor dialog box.
Designate the sample name, experiment type (ABS), ddPCR Supermix for Probes (No dUTP) as the supermix type, target name(s), and target type(s): Ch1 for FAM and Ch2 for HEX or VIC.
Select Apply to load the wells and, when finished, select OK.
Once the plate layout is complete, select Run to begin the droplet reading process. Select the appropriate dye set used and run options when prompted.
After data acquisition (~80s per well), select samples in the well selector under Analyze. Examine the automatic thresholding applied to the 1-D or 2-D amplitude data and, if necessary, set thresholds or clusters manually.
The concentration reported is copies per uL of RNA in the final 1x ddPCR reaction.