Concentration and nucleic acid extraction of viruses from wastewater primary sludge

Prepare a 1% volume Tween 20 stock solution. For a 500mL stock, combine 5mL of Tween 20 with 495mL of 1X PBS. Filter sterilize with a 0.22 um vacuum filtration unit.

Transfer sludge sample (within secondary container) from the refrigerator to the fume hood. Remove the sample bottle from the secondary container and unseal the bottle by removing the affixed Parafilm.

Prepare aliquots of sludge. Invert the bottle of sludge several times to re-suspend contents, then carefully open it. Using a fresh 50 mL serological pipette, aspirate and dispense 20mL of sludge into a 50 mL centrifuge tube. Repeat for desired number of sample replicates. For a negative control, add 20mL of 1X PBS to a centrifuge tube using a fresh 50 mL serological pipette.

Add 20mL of 1% (v/v) Tween 20 to each centrifuge tube (including controls) for a final concentration of 0.5% (v/v) Tween 20.

Cap and parafilm all bottles and centrifuge tubes.

Place all centrifuge tubes on a vortexer using a 50 mL tube adapter. Shake for 1000rpm

Transfer all centrifuge tubes from the vortexer to a sonication bath, and sonicate for 0h 1m 0s at 40 kHz. Use a paper towel to dry the tubes when finished.

Transfer all centrifuge tubes from the fume hood to a centrifuge equipped with the appropriate rotor and/or adapters (ex: Beckman Coulter Avanti J series with JA-14.50 rotor) . Centrifuge the samples at 10000rpm,4°C. After centrifugation is complete, wait 0h 10m 0s (as recommended by EHS) to allow aerosols to settle before opening the centrifuge. Remove samples from the centrifuge and return them to the fume hood.

In the fume hood, prepare a separate 0.45 um vacuum filtration apparatus for each sample and control by attaching the filtration unit to a clean pyrex bottle.

Decant the supernatant from the sludge sample centrifuge tube directly into a 0.45 um vacuum filtration top attached to a sample collection bottle, taking care not to dislodge the pellet.

Begin vacuum filtration by capping the vacuum filtration top and opening the vacuum line. When complete, cap the pyrex bottle and set aside. Repeat for each sample replicate.

For the negative control centrifuge tube, decant directly into a 0.45 um vacuum filtration top. There should not be a pellet. Perform vacuum filtration as was done for the sludge, cap the pyrex bottle, and set aside.

Perform the "Start-up" protocol for the InnovaPrep Concentrating Pipette Select.

• Turn on the Concentrating Pipette, and navigate to "Maintenance" and then "Start-up". Follow the prompts.
• Check that the maintenance tip is in place.
• Place the waste line in the proper position.
• Remove the storage fluid line and insert the foam elution canister.
• Ensure that the screen reads "WWULTRA".

Run the concentration protocol for the filtered sludge sample (ie, filtrate).

Remove the maintenance tip and place a fresh Ultra CPT into the tip port. Lower the tip into the sample.

Press "Start Run", allow the Concentrating Pipette to aspirate the waste, and wait until it beeps to signal the end of the run.

While holding a 5 mL centrifuge tube under the Tip, press "Elute" and catch the foam that is dispensed.

After the foam degasses, add 400µL of the Zymo DNA/RNA shield reagent.

Run the concentration protocol for the negative control. Perform as is done for the sludge sample.

Incubate samples with added Zymo DNA/RNA shield reagent at room temperature for 0h 30m 0s. If samples are sitting for longer then store at 4°C

Perform the "Shut Down" protocol for the InnovaPrep Concentrating Pipette Select.

• Navigate to "Maintenance" and then "Shut Down".
• Place the maintenance tip into the tip port.
• Remove the elution canister.
• Check to ensure that there is adequate storage fluid and insert the storage fluid line.
• Turn off the device and remove the waste line.

Gather the materials and reagents for the Zymo quick-DNA/RNA Viral Kit in the Biosafety Cabinet. Equilibrate samples to room temperature.

Add 1600µL of Viral DNA/RNA buffer to the sample (a 2:1 ratio) and vortex briefly to mix.

Transfer up to 700µL of lysate into a Zymo-SpinTM IIC-XLR Column in a collection Tube and centrifuge for 12.000x g. Discard the flow-through in the miniprep waste container.

Repeat until full lysate volume is processed.

Add 500µL of Viral Wash Buffer to the column, centrifuge for 12.000x g and discard the flow-through.

Repeat the previous step.

Add 500µL ethanol (95-100%) to the column and centrifuge for 12000x g to ensure complete removal of the wash buffer.

Transfer the column to a clean collection tube, and centrifuge at 12000x g to remove any remaining ETOH.

Carefully, transfer the column into a 1.5 mL nuclease-free tube.

Add 50µL DNase/RNase-Free Water directly to the column matrix and incubate at RT for 0h 1m 0s. Centrifuge for 12000x g to collect the eluate.

Make desired number of aliquots for storage. Place all extracted nucleic acids in a freezer set to -80°C