Mar 01, 2024
Version 2
Concentration and nucleic acid extraction of viruses from wastewater influent V.2
- Ari N Machtinger1,2,
- Olivia S Hershey1,2,
- William J Bradshaw1,2,
- Daniel P Rice1,2,
- Michael R McLaren1,2
- 1Media Laboratory, Massachusetts Institute of Technology;
- 2SecureBio
External link: https://naobservatory.org
Protocol Citation: Ari N Machtinger, Olivia S Hershey, William J Bradshaw, Daniel P Rice, Michael R McLaren 2024. Concentration and nucleic acid extraction of viruses from wastewater influent. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko1rwv5r/v2Version created by Michael R McLaren
Manuscript citation:
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 20, 2024
Last Modified: March 15, 2024
Protocol Integer ID: 93857
Keywords: wastewater-based epidemiology, WBE, viral concentration, metagenomics, viromics, wastewater, wastewater influent
Funders Acknowledgement:
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 influent for the purposes of untargeted RNA and DNA sequencing of viruses present in wastewater.
Protocol overview: 200 mL of raw influent wastewater 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 pellet 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 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)
- 2 x 250 mL pyrex bottles
- 2 x 5 mL centrifuge tubes
- 2 x 50 mL serological pipettes (VWR No. 170358N)
- 1.5 mL microcentrifuge tubes (VWR No. 1420-2600)
- PREempt wipes (VWR No. 10822-456)
- Parafilm (Millipore Sigma No. HS234526C)
- RNaseZap
- Kimwipes (VWR No. 34120)
- Paper towels
- Filtered micropipette tips
Reagents:
- Tween 20
- 1x Phosphate Buffered Saline (w/o Ca & Mg)
- Ice
- All buffers in the Zymo quick-DNA/RNA Viral Kit
Equipment:
- Bransonic M 1800 Sonicator (filled ¾ with tap water)
- Attachment for vortexer (Cole-Parmer No. UX-04724-89)
- Scientific Industries Vortex Genie 2
- Vacuum Line
- Micropipettes (1000 uL, 200 uL, 10 uL, 2 uL, 1 uL) and holder
- Timer
- Floor Centrifuge (Ex: Beckman Coulter Avanti J series)
- Rotor compatible with 50 mL tubes (Ex: Beckman Coulter JA-14.50)
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, sterile-filtered 10% Tween 20, PBS). Label centrifuge tubes (five tubes for each influent sample, and two for a negative control sample). Ensure proper PPE.
Reagent Preparation
Reagent Preparation
7m
7m
Prepare a 10 % volume Tween 20 stock solution. For a 40 mL stock, combine 4 mL of Tween 20 with 36 mL of 1X PBS. Filter sterilize with a 0.22 um vacuum filtration unit.
Expected result
This recipe produces enough 10% Tween 20/PBS solution for approximately 100 dissociation treatments.
7m
Part 1: Influent Handling, Dissociation, Centrifugation, Filtration
Part 1: Influent Handling, Dissociation, Centrifugation, Filtration
1h
1h
Safety information
Refer to the 'Safety Warnings' section for biosafety precautions necessary for handling raw wastewater samples.
Transfer influent 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 influent. Invert the bottle of influent several times to re-suspend contents, then carefully open it. Using a fresh 50 mL serological pipette, aspirate and dispense 40 mL of influent into a 50 mL centrifuge tube. Repeat until 200 mL of influent has been dispensed across 5 centrifuge tubes. Repeat for desired number of sample replicates. For a negative control, add 40 mL of 1X PBS to two centrifuge tubes each using a fresh 50 mL serological pipette, for a total volume of 80 mL .
Note
The 200 mL sample is separated into five 50 mL tubes due to supply and equipment limitations.
EHS also recommended limiting sample volume to 40 mL in each tube to reduce the risk of leaks.
10m
Add 400 µL of 10 % (v/v) Tween 20 stock solution to each centrifuge tube for a final concentration of 0.1 % (v/v) Tween 20.
3m
Cap and parafilm all bottles and centrifuge tubes.
Safety information
Return the influent bottle to secondary containment, wipe exposed surfaces with pre-empt wipes, and return the remaining influent to refrigeration.
5m
Place all centrifuge tubes on a vortexer using a 50 mL tube adapter. Shake for 1000 rpm, 00:01:00
2m
Transfer all centrifuge tubes from the vortexer to a sonication bath, and sonicate for 00:01:00 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 10000 rpm, 4°C, 00:05:00 . After centrifugation is complete, wait 00:10:00 (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.
Note
The centrifuge tubes for each sample will be combined during this step. For example:
- The contents of the five influent tubes (~ 200 mL ) will be combined using one filtration unit
- The contents of the two control tubes (~ 80 mL ) will be combined into the second filtration unit.
5m
Decant the supernatant from all five influent tubes directly into a 0.45 um vacuum filtration top attached to the influent sample collection bottle, taking care not to dislodge the pellet.
Centrifuge tubes contain a total of 200 mL influent sample (40 mL x 5 tubes). The total volume of the sample is filtered and collected in the bottle.
2m
Begin vacuum filtration by capping the vacuum filtration top and opening the vacuum line. When complete, cap the pyrex bottle and set aside.
Expected result
The filtrate volume will vary depending on the amount of solid material in the sample.
5m
For the negative control centrifuge tubes, decant both directly into a 0.45 um vacuum filtration top. There should not be a pellet. Perform vacuum filtration as was done for the influent, cap the pyrex bottle, and set aside.
2m
Part 2: Concentration via InnovaPrep Concentrating Pipette Select
Part 2: Concentration via InnovaPrep Concentrating Pipette Select
1h 10m
1h 10m
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 influent sample.
25m
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 17 min, but can vary based on the consistency of the sample.
20m
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 400 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 in the same manner as the influent sample.
Note
Make sure to use a fresh Tip.
5m
Incubate samples with added Zymo DNA/RNA shield reagent at room temperature for 00:30:00 . If samples are sitting for longer then store at 4 °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
Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
20m
20m
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 1600 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 700 µL of lysate into a Zymo-SpinTM IIC-XLR Column in a collection Tube and centrifuge for 12.000 x g, 00:02:00 . Discard the flow-through in the miniprep waste container.
Repeat until full lysate volume is processed.
8m
Add 500 µL of Viral Wash Buffer to the column, centrifuge for 12.000 x g, 00:00:30 and discard the flow-through.
30s
Repeat the previous step.
2m
Add 500 µL ethanol (95-100%) to the column and centrifuge for 12000 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 12.000°C, 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 50 µL DNase/RNase-Free Water directly to the column matrix and incubate at RT for 00:01:00 . Centrifuge for 12000 x g, 00:00:30 to collect the eluate.
1m 30s
Place all extracted nucleic acids in a freezer set to -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