TITLE: Concentration of viruses from wastewater influent using organic flocculation (PEG)
AUTHORS: Katy Graham, Marlene Wolfe, Stephanie Loeb, Alexandria Boehm, Krista Wigginton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in influent wastewater samples from wastewater treatment plants. PEG precipitation is used to concentrate viruses in the samples. The concentrated pellet is recovered and resuspended to be utilized for the next steps of the protocol. Bovine coronavirus is used as a recovery control for the concentration step. The RNA extraction is acheived using a commercial kit and an inhibitor removal kit is used to remove inhibitors that were co-extracted with the RNA. The RNA from this protocol can be used in droplet digital RT-PCR assays. A protocol for the dd-RTPCR assays is available from our group. </div><div class = "text-block"><h2>Biosafety Concerns</h2></div><div class = "text-block">Concentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines. </div></div>

[STEPS]
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Remove chosen samples from freezer storage, and thaw at 4°C. This should take about five hours for a 50 mL conical tube, and samples should not be placed at 4°C longer than 24 hrs prior to processing. Record the sample’s volume.
Follow safe biosafety practices throughout. Samples should never be opened outside the biosafety cabinet (BSC). Bring the centrifuge rotor to the BSC before opening.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Load samples into centrifuge rotor, ensuring that samples are balanced. Centrifuge at to remove suspended solids.
Centrifuge: 24000 34, 15 min, 4 10
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Remove rotor from centrifuge and open it in the BSC. Pipette off about 40mL of supernatant, avoiding the solid pellet, into a new 50mL falcon tube.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Add / PEG8000 and  NaCl to each sample and shake to mix. The PEG8000 may clump together, so gentle vortexing may be applied.
8 g
100 mL
In order to determine the correct mass of PEG8000 and NaCl to add, calculate mass based on the sample volume.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Incubate sample at  overnight.
4 °C
If it is important to complete concentration in a single day incubation may be shortened to no less than 4 hours, however overnight incubation produces the best results.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Individually spike each sample with  BCoV stock. Pipet the spike directly into the samples. Gently vortex to mix and allow to equilibrate  for .
75 µl
on ice
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Remove sample from 4°C the next morning. Load samples into centrifuge rotor, ensuring that samples are balanced. Centrifuge at .
Centrifuge: 20000 34, 30 min, 4 10
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Remove samples from the rotor, and remove and discard ~20mL of sample. Wash the sides of the tube with the remaining supernatant.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Centrifuge again at .
Centrifuge: 20000 34, 20 min
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
?. [Extract RNA: 5 hours per 20 samples]
RNA extraction is performed using the Qiagen AllPrep PowerViral Kit. Before starting:Prepare Solution PM1/β-ME: Warm Solution PM1 at  for  prior to use to dissolve precipitates. Use Solution PM1 while still warm. Shake to mix before using.Add β-ME to Solution PM1 to produce a mixture of Solution PM1/β-ME with a final β-ME concentration of 10 μl/ml ( of β-ME with  warm PM1 per sample). The mixture of Solution PM1/β-ME loses its effectiveness over time, so prepare a fresh batch each time you use the kit. You will need of the Solution PM1/β-ME mixture per prep.
55 °C
6 µl
594 µl
600 µl
?. [Extract RNA: 5 hours per 20 samples]
Pipet of viral concentrate into a PowerBead Tube, Glass 0.1 mm. Include one extraction blank per set of extractions.
200 µl
?. [Extract RNA: 5 hours per 20 samples]
Add  of the Solution PM1/β-ME mixture to the PowerBead Tube.
600 µl
?. [Extract RNA: 5 hours per 20 samples]
Secure the bead tubes horizontally to a Vortex Adapter (cat. no. 13000-V1-24). The tube caps should be pointing toward the center of the adapter. Vortex at maximum speed for in the BSC.
?. [Extract RNA: 5 hours per 20 samples]
Centrifuge at  at room temperature. Transfer the supernatant to a clean 2 ml Collection Tube.
Centrifuge: 13000 34, 1 min
Stick the pipet tip down into the beads to pipet out all of the supernatant- it is okay if you get some beads because they will be centrifuged into a pellet in the following steps.
?. [Extract RNA: 5 hours per 20 samples]
Add  of Solution IRS and vortex briefly to mix. Incubate at  or on ice for .
200 µl
4 °C
IRS is the inhibitor removal solution- this is a really important step!
?. [Extract RNA: 5 hours per 20 samples]
Centrifuge at  . Avoiding the pellet, transfer the supernatant to a clean 2.2 ml  Collection Tube. Do not transfer more than .
Centrifuge: 13000 34, 1 min
700 µl
If you have access to a QIAcube, this is where the QIAcube Connect protocol begins.
?. [Extract RNA: 5 hours per 20 samples]
Add  each of Solution PM3 and Solution PM4 to each tube. Vortex briefly to mix.
600 µl
?. [Extract RNA: 5 hours per 20 samples]
Load of supernatant into an MB Spin Column and centrifuge at  . Discard the flow through and repeat until all the supernatant has been loaded onto the MB Spin Column.
625 µl
Centrifuge: 13000 34, 1 min
?. [Extract RNA: 5 hours per 20 samples]
Shake to mix Solution PM5 and add to the MB Spin Column. Centrifuge at .
600 µl
Centrifuge: 13000 34, 1 min
?. [Extract RNA: 5 hours per 20 samples]
Discard flow-through. Add  of Solution PM4. Centrifuge at  .
600 µl
Centrifuge: 13000 34, 1 min
?. [Extract RNA: 5 hours per 20 samples]
Discard flow-through and Centrifuge at   to remove residual buffer.
Centrifuge: 13000 34, 2 min
?. [Extract RNA: 5 hours per 20 samples]
Place the MB Spin Column in a clean 2 ml Collection Tube.
?. [Extract RNA: 5 hours per 20 samples]
Add  of RNase-Free Water to the center of the MB Spin Column membrane. Incubate for  at room temperature. Pipet directly onto white membrane using a REACH pipet tip if needed.
50 µl
?. [Extract RNA: 5 hours per 20 samples]
Centrifuge at  . Discard the MB Spin Column. The DNA/RNA is now ready for downstream applications. Proceed with inhibitor removal immediately before storing DNA/RNA for future use.
Centrifuge: 13000 34, 1 min
?. [Inhibitor Removal: 1 hour per 24 samples]
Place one column into one collection tube for each sample you need and add  of Prep solution to each column.
600 µl
?. [Inhibitor Removal: 1 hour per 24 samples]
Spin at ; discard collection tube with flow through.
Centrifuge: 8000 34, 3 min
?. [Inhibitor Removal: 1 hour per 24 samples]
Put column into a labeled 1.5mL LoBind tube.
?. [Inhibitor Removal: 1 hour per 24 samples]
Add ~ of RNA/DNA extract to each column.
50 µl
?. [Inhibitor Removal: 1 hour per 24 samples]
Spin at .
Centrifuge: 16000 34, 3 min
?. [Inhibitor Removal: 1 hour per 24 samples]
Use cleaned extract for Nanodrop, Qubit, or RT, and store at until analysis.
-80 °C
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Resuspend the pellet in a total volume of , using 1x PBS to supplement any liquid remaining in the tube.
200 µl
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Gently vortex to resuspend the pellet.
?. [Concentrate: 3 hours on day 1 and 1 hour on day 2 for 8 samples]
Transfer the resuspended pellet to a 1mL cryotube and store at .
-80 °C