Mar 02, 2024

Public workspaceConcentration and nucleic acid extraction of viruses from wastewater primary sludge

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq3k1xlk5/v1
Concentration and nucleic acid extraction of viruses from wastewater primary sludge
  • Ari Machtinger1,2,
  • Olivia Hershey1,2,
  • William J Bradshaw1,2,
  • Emily H Soice1,2,
  • Daniel P Rice1,2,
  • Michael McLaren1,2
  • 1Media Laboratory, Massachusetts Institute of Technology;
  • 2SecureBio
  • SecureBio Detection
Michael R McLaren
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgq3k1xlk5/v1
External link: https://naobservatory.org
Protocol Citation: Ari Machtinger, Olivia Hershey, William J Bradshaw, Emily H Soice, Daniel P Rice, Michael McLaren 2024. Concentration and nucleic acid extraction of viruses from wastewater primary sludge. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3k1xlk5/v1
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: January 18, 2024
Last Modified: March 15, 2024
Protocol Integer ID: 93742
Keywords: wastewater-based epidemiology, WBE, viral concentration, metagenomics, viromics, wastewater, wastewater sludge, total nucleic acid extraction from wastewater, nucleic acid extraction of virus, total nucleic acid extraction, nucleic acid extraction, rna viral kit, wastewater primary sludge this protocol detail, wastewater processing protocol, dna sequencing of virus, modified wastewater processing protocol, wastewater sample, viral particles from solid, ml of wastewater sludge, wastewater sludge, viral particle, wastewater, primary sludge, present in wastewater, wastewater matrix, nucleic acid, extraction, pes 75 mm filtration unit, untargeted rna, acid extraction, dna sequencing, purposes of untargeted rna, rna, mm filtration unit, filtrate, concentrated product, innovaprep concentrating pipette select, performing concentration, virus, pellet, processing
Funders Acknowledgements:
Open Philanthropy
Grant ID: NA
Musk Foundation
Grant ID: NA
Abstract
This protocol details our workflow for performing concentration and total nucleic acid extraction from wastewater primary sludge (settled solids) for the purposes of untargeted RNA and DNA sequencing of viruses present in wastewater.

Protocol overview: In this protocol, 20 mL of wastewater sludge is concentrated to a final volume of 400 uL using the InnovaPrep Concentrating Pipette Select. Prior to concentration, the wastewater sample is treated with Tween 20 and sonicated to dissociate viral particles from solids in the wastewater matrix. The sample is then centrifuged to remove larger solids. The pellet is discarded, and the supernatant is filtered with a 0.45 um PES 75 mm filtration unit to remove remaining suspended solids and bacteria. This filtrate is then concentrated with the Concentrating Pipette, using Ultra CPT tips and recommended device settings for the InnovaPrep modified wastewater processing protocol. Nucleic acids are then extracted from the concentrated product using the Zymo quick-DNA/RNA Viral kit using the manufacturer protocol with a few modifications that we have found to be helpful.
Guidelines
RNA processing and handling: Please review Protocol Note: Working with RNA Samples before handling RNA samples.
Materials
Materials:
(for one sample replicate and one negative control)
Reagents:
Equipment:

Safety warnings
Biosafety precautions: All raw wastewater samples will be received and stored with primary and secondary containment. The primary container (the bottle or falcon tube) should remain in the secondary container (a Ziploc bag containing paper towels to absorb spills) until processed. All raw samples must be handled within a dedicated fume hood or biosafety cabinet. All laboratory personnel handling these samples must use safety glasses, gloves, and lab coats. Samples will be transported between processing stations within a secondary container that has been cleaned with PreEmpt. All surfaces (outside of the fume hood/sash, centrifuge lid and rotor, etc.) will also be wiped down with PreEmpt. All autoclave-able bottles that are in contact with wastewater samples will be cleaned by filling with 10% bleach for at least 20 minutes before disposal.
Before start
Read the 'Safety Warnings' section for biosafety precautions necessary for handling raw wastewater samples. Prepare the fume hood for wastewater handling, gather materials and reagents (centrifuge tubes, serological pipettes, pipette tips, micropipettes, a marker, strips of parafilm, PBS). Label centrifuge tubes (one for each influent sample, and two for a negative control sample). Ensure proper PPE.
Reagent Preparation
7m
Prepare a Concentration1 % volume Tween 20 stock solution. For a Amount500 mL stock, combine Amount5 mL of Tween 20 with Amount495 mL of 1X PBS. Filter sterilize with a 0.22 um vacuum filtration unit.
Expected result
This recipe produces enough 1% Tween 20/PBS solution for approximately 24 dissociation treatments.

7m
Part 1: Sludge Handling, Dissociation, Centrifugation, Filtration
52m
Safety information
Refer to the 'Safety Warnings' section for biosafety precautions necessary for handling raw wastewater samples.

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.
5m
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 Amount20 mL of sludge into a 50 mL centrifuge tube. Repeat for desired number of sample replicates. For a negative control, add Amount20 mL of 1X PBS to a centrifuge tube using a fresh 50 mL serological pipette.

Note
EHS recommended limiting sample volume to 40 mL in each tube to reduce the risk of leaks.

10m
Add Amount20 mL of Concentration1 % (v/v) Tween 20 to each centrifuge tube (including controls) for a final concentration of Concentration0.5 % (v/v) Tween 20.

3m
Cap and parafilm all bottles and centrifuge tubes.
Safety information
Return the sludge bottle to secondary containment, wipe exposed surfaces with pre-empt wipes, and return the remaining sludge to refrigeration.

5m
Place all centrifuge tubes on a vortexer using a 50 mL tube adapter. Shake for Shaker1000 rpm, 00:01:00

2m
Transfer all centrifuge tubes from the vortexer to a sonication bath, and sonicate for Duration00:01:00 at 40 kHz. Use a paper towel to dry the tubes when finished.
1m
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 Centrifigation10000 rpm, 4°C, 00:05:00 . After centrifugation is complete, wait Duration00:10:00 (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.
15m
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.
5m
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.
The centrifuge tube contains 40 mL of diluted sludge sample. The total volume of the sample is filtered and collected in the bottle.

1m
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.


Expected result
The filtrate volume will vary depending on the amount of solid material in the sample.

3m
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.
2m
Part 2: Concentration via InnovaPrep Concentrating Pipette Select
55m
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".
5m
Run the concentration protocol for the filtered sludge sample (ie, filtrate).
10m
Remove the maintenance tip and place a fresh Ultra CPT into the tip port. Lower the tip into the sample.
Note
Ensure that the Tip is as close to the bottom of the sample bottle as possible. If necessary, the bottle can be balanced on its edge while a weighted object holds down the top of the Concentrating pipette. A bottle of PBS can be used as the weighted object.

1m
Press "Start Run", allow the Concentrating Pipette to aspirate the waste, and wait until it beeps to signal the end of the run.
Note
A timer will run on the display during the concentration. The Concentrating Pipette will stop aspirating and begin to beep when the Tip detects air instead of liquid sample. Concentration should take roughly 5 min, but can vary based on the consistency of the sample.

5m
While holding a 5 mL centrifuge tube under the Tip, press "Elute" and catch the foam that is dispensed.
2m
After the foam degasses, add Amount400 µL of the Zymo DNA/RNA shield reagent.
Expected result
The eluate should be roughly 400 uL. The Zymo DNA/RNA shield reagent is added at a 1:1 ratio of sample:reagent.

2m
Run the concentration protocol for the negative control. Perform as is done for the sludge sample.
Note
Make sure to use a fresh Tip for each sample.

5m
Incubate samples with added Zymo DNA/RNA shield reagent at room temperature for Duration00:30:00 . If samples are sitting for longer then store at Temperature4 °C

30m
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.
5m
Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
6m
Gather the materials and reagents for the Zymo quick-DNA/RNA Viral Kit in the Biosafety Cabinet. Equilibrate samples to room temperature.
Note
Refer to the "Guidelines" section for instructions on processing and handling RNA samples.

5m
Add Amount1600 µL of Viral DNA/RNA buffer to the sample (a 2:1 ratio) and vortex briefly to mix.

Note
The volume is scaled according to the manufacturers recommendation.

3m
Transfer up to Amount700 µL of lysate into a Zymo-SpinTM IIC-XLR Column in a collection Tube and centrifuge for Centrifigation12.000 x g, 00:02:00 . Discard the flow-through in the miniprep waste container.
Repeat until full lysate volume is processed.
8m
Add Amount500 µL of Viral Wash Buffer to the column, centrifuge for Centrifigation12.000 x g, 00:00:30 and discard the flow-through.

30s
Repeat the previous step.
2m
Add Amount500 µL ethanol (95-100%) to the column and centrifuge for Centrifigation12000 x g, 00:01:00 to ensure complete removal of the wash buffer.

1m
Transfer the column to a clean collection tube, and centrifuge at Centrifigation12000 x g, 00:01:00 to remove any remaining ETOH.
Note
This step is not included in the Zymo manual, but reduces the chance of ETOH carryover and subsequent inhibition of downstream enzymatic steps.

1m
Carefully, transfer the column into a 1.5 mL nuclease-free tube.
1m
Add Amount50 µL DNase/RNase-Free Water directly to the column matrix and incubate at RT for Duration00:01:00 . Centrifuge for Centrifigation12000 x g, 00:00:30 to collect the eluate.
1m 30s
Make desired number of aliquots for storage. Place all extracted nucleic acids in a freezer set to Temperature-80 °C

Note
Optional: Perform nucleic acid quantification prior to freezing. Perform RNA or DNA quantification using the Qubit HS RNA assay kit or the Qubit 1X dsDNA Assay kit.

3m
Protocol references