TITLE: Wastewater Concentration by Adsorption and Direct Extraction for SARS-CoV-2 RNA Detection and Quantification using RT-ddPCR
AUTHORS: Aaron Bivins, Warish Ahmed, Devin North, Kyle Bibby

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 </span><span style = "font-style:italic;">et al</span><span>. 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.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/e26be990317291a29f5b9a47f7ae4a76205545798e28b5a05529a22b922c499b/cbxn8v4e.png" /></div></div>

[STEPS]
?. [Sample Collection]
Collect a   sample of primary influent or raw wastewater (grab sample or composite) in a sterile sample collection bottle. Maintain the sample at   during transport to the lab.
1 L
4 °C
?. [Sample Collection]
Upon sample receipt at the laboratory spike  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   for  . 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.
1 mL
4 °C
?. [Sample Collection]
If sample is to be concentrated immediately temporary storage at  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  until concentration is to be completed.  Aliquot  of the wastewater sample for concentration as described below and store the remaining  at  for any future anayses that may be required.
4 °C
-80 °C
500 mL
500 mL
-80 °C
?. [HA Concentration]
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.
?. [HA Concentration]
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.
?. [HA Concentration]
Using 2N Hydrochloric acid adjust the pH of each   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.
500 mL
?. [HA Concentration]
Slowly add  of wastewater sample to a single filtration assembly funnel top in increments of approximately   . Allow the suspended settles to settle. Add the remaining   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  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.
250 mL
50 mL
250 mL
250 mL
?. [HA Concentration]
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.
?. [HA Concentration]
Store filters in PowerBead tubes at  until extraction.
-80 °C
?. [RNA Extraction]
RNA Extraction is performed using the Qiagen Allprep PowerViral DNA/RNA Kit with modifications.
?. [RNA Extraction]
Sample Homogenization: Briefly centrifuge each PowerBead tube ~ then add  of Warm Solution PM1 and  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 ~.  After the fourth and final homogenization cycle, centrifuge the PowerBead tubes at  at room temperature. Transfer   of the resulting supernatant to a clean 2 mL Collection Tube.
Centrifuge: 5000 34
650 µl
6.5 µl
Centrifuge: 5000 34
Centrifuge: 13000 34, 1 min
450 µl
?. [RNA Extraction]
Add   of Solution IRS and vortex briefly. Incubate at  for  .
150 µl
4 °C
?. [RNA Extraction]
Centrifuge the 2 mL Collection Tube at  . Transfer  of supernatant to a clean 2.2 mL Collection Tube avoiding the pelleted material.
Centrifuge: 13000 34, 1 min
500 µl
?. [RNA Extraction]
Add  of Solution PM3 and   of Solution PM4 to each 2.2 mL Collection Tube and vortex briefly to mix.
600 µl
600 µl
?. [RNA Extraction]
Load   of mixture onto an MB Spin Column and centrifuge at  . Discare the flow through and repeat until all the supernatant has been filtered through the Spin Column.
625 µl
Centrifuge: 13000 34, 1 min
?. [RNA Extraction]
Shake to mix Solution PM5 and add  to the MB Spin Column. Centrifuge at  .
600 µl
Centrifuge: 13000 34, 1 min
?. [RNA Extraction]
Discard the flow through and add  of Solution PM4. Centrifuge at  . Discard the flow through and centrifuge at  .
600 µl
Centrifuge: 13000 34, 1 min
Centrifuge: 13000 34, 2 min
?. [RNA Extraction]
Place the MB Spin Column into a clean 2 mL Collection Tube. Add  of RNase-free water to the center of the white column membrane and incubate for at least  . Centrifuge at  and discard the MB Spin Column.
80 µl
Centrifuge: 13000 34, 1 min
?. [RNA Extraction]
Spin the 2 mL Collection Tube at  and transfer  of supernatant to a 1.5 mL Eppendorft LoBind Tube.
Centrifuge: 13000 34, 2 min
60 µl
?. [RNA Extraction]
The DNA/RNA is now ready for downstream analaysis. Store at  until further analysis.
-80 °C
?. [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.
?. [RT-ddPCR Oligos]
Primers, probes, and control materials:Unless otherwise specified, all oligonucleotide materials are purchased from Integrated DNA Technologies (IDT, Corallvile, IA).
?. [RT-ddPCR Oligos]
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-actinAmplicon length: 124 bp  AB1FWD Primer5' GCA ATG CTG CAG GAC TAG GTA TAA T 3'2REV Primer5' ACA CTG TAA TTG ATG ACC CCA TTC T 3'3Probe/5HEX/AC CAA GAC T/ZEN/T GTA TGA TGC TGC CAA AGC A/3IABkFQ/
AB1FWD Primer5' GCA ATG CTG CAG GAC TAG GTA TAA T 3'2REV Primer5' ACA CTG TAA TTG ATG ACC CCA TTC T 3'3Probe/5HEX/AC CAA GAC T/ZEN/T GTA TGA TGC TGC CAA AGC A/3IABkFQ/
?. [RT-ddPCR Oligos]
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).The N gene control plasmid is also avialable from IDT.
?. [RT-ddPCR]
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:   x 2(2) Reverse Transcriptase, RT:   x 2(3) Dithiothreitol, DTT solution:   x 2All components on the One-Step RT-ddPCR Advanced Kit for Probes are stable for 12 months when stored at  . Repeated freezing and thawing of the supermix is not recommended. DTT should be aliquoted to multiple tubes and stored at  to minimize freezing and thawing.
500 µl
200 µl
1 mL
-20 °C
-20 °C
?. [RT-ddPCR]
Thaw all components on ice for  . Mix each component thoroughly by vortexing each tube several times for   to ensure homogeneity because a concentration gradient may form during  storage. Centrifuge briefly to collect contents at the bottom of each tube.
-20 °C
?. [RT-ddPCR]
Prepare samples at the desired concentration before setting up the reaction mix. Store samples at  while preparing the reaction mix.
4 °C
?. [RT-ddPCR]
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                                    Supermix          4x                                                      1x                                     RT                      n/a                                                    n/a                                    DT        Probe  Fwd    Rev     WW RNA        Variable*                                          Variable*                               Total Volume per reaction:                                                                                 N1 or N2 reagent volumes per reaction:Reagent               Stock Conc.                                Reaction Conc.           Volume per reaction (uL)Water                        n/a                                                    n/a                                    Supermix                  4x                                                      1x                                     RT                              n/a                                                    n/a                                    DT                IDT PreMix        Probe @ 250 nM                                                                                                         Primers @ 1000 nMWW RNA                Variable*                                          Variable*                              Total Volume per reaction:                                                                                         *Suggested input quantities of total RNA are 100 fg - 100 ng per reaction
5.59 µl
5.25 µl
2.1 µl
1.05 µl
0.055 µl
1.98 µl
1.98 µl
4 µl
22 µl
6.45 µl
5.25 µl
2.1 µl
1.05 µl
3.15 µl
4 µl
22 µl
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
Once the reaction mixtures are ready, place a DG8 Cartridge into a DG8 Cartridge Holder. Next load  of each reaction mix into the sample well of a DG8 Cartridge for QX200/QX100 Droplet Generator followed by  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.
20 µl
70 µl
?. [RT-ddPCR]
Load the DG8 Cartridge Holder onto the QX200 Droplet Generator and close the lid to begin droplet generation.
?. [RT-ddPCR]
Once droplet generation is complete, carefully transfer  of the droplet oil emulsion from the DG8 Cartridge to a 96-well ddPCR plate.
40 µl
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
After sealing the 96-well plate, incubate the plate at   for  .
4 °C
?. [RT-ddPCR]
Place the sealed 96-well plate on the C1000 Touch Thermal Cycler and perform thermal cycling as specificed in Table 2 below.Cycling Step                        Temperature (C)                Time                        Number of CyclesReverse Transcription                                             1Enzyme Activation                                                   1Denaturation                                                           40xAnnealing/Extension                                             above Enzyme Deactivation                                                1Hold                                                               Infinite                                         1
50 °C
95 °C
95 °C
59 °C
98 °C
4 °C
?. [RT-ddPCR]
When thermal cycling is complete, allow the 96-well plate to incubate at   for  .
4 °C
?. [RT-ddPCR]
Place the 96-well plate in the QX200 Droplet Reader and ensure there is adequate ddPCR Droplet Reader Oil in the reservoir.
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
Under Setup, double click on a well in the plate layout to open the Well Editor dialog box.
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
Select Apply to load the wells and, when finished, select OK.
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
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.
?. [RT-ddPCR]
The concentration reported is copies per uL of RNA in the final 1x ddPCR reaction.