Concentration and nucleic acid extraction of viruses from aggregated airplane waste

Ari N Machtinger, Olivia S Hershey, William J Bradshaw, Daniel P Rice, Michael R McLaren

Published: 2024-03-03 DOI: 10.17504/protocols.io.5jyl8poy6g2w/v1

Abstract

This protocol details our workflow for performing concentration and total nucleic acid extraction from aggregated airplane waste for the purposes of untargeted RNA and DNA sequencing of viruses present in wastewater.

Protocol overview: In this protocol, 40 mL of aggregated airplane waste is concentrated to a final volume of 400 uL using the InnovaPrep Concentrating Pipette Select. Prior to concentration, the waste sample is treated with Tween 20 and sonicated to dissociate viral particles from solids in the wastewater matrix. The waste sample is filtered using a 0.45 um PES 75 mm filtration unit, with an added pre-filter (2um glass fiber) to remove 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.

Before start

Read the 'Safety Warnings' section for biosafety precautions necessary for handling raw airplane waste 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 (two for each influent sample, and two for a negative control sample). Ensure proper PPE.

Steps

Reagent Preparation

Prepare a 10% volume Tween 20 stock solution. For a 40mL stock, combine 4mL of Tween 20 with 36mL of 1X PBS. Filter sterilize with a 0.22 um vacuum filtration unit.

Citation

This recipe produces enough 10% Tween 20/PBS solution for approximately 20 dissociation treatments.

Part 1: Waste Handling, Dissociation, Filtration

Safety information

Refer to the 'Safety Warnings' section for biosafety precautions necessary for handling raw wastewater samples.

Transfer the waste 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 aggregated airplane waste. Invert the bottle of waste several times to re-suspend contents, then carefully open it. Using a fresh 50 mL serological pipette, aspirate and dispense 20mL of waste into two 50 mL centrifuge tubes each. Repeat for desired number of sample replicates. For a negative control, add 20mL of 1X PBS to two centrifuge tubes each using a fresh 50 mL serological pipette, for a total volume of 40mL.

Note

The 40 mL sample is separated into two 50 mL tubes, each containing 20 mL of airplane waste and 20 mL of dilution and dissociation reagent (step 4).Additionally, EHS recommended limiting sample volume to 40 mL in each tube to reduce the risk of leaks.

Add 18mL of 1X PBS to each centrifuge tube. 2 mL of 10% volume Tween 20 stock solution to each centrifuge tube for a final concentration of 5% (v/v) Tween 20.

Cap and parafilm all bottles and centrifuge tubes.

Safety information

Return the waste bottle to secondary containment, wipe exposed surfaces with pre-empt wipes, and return the remaining waste to refrigeration.

Place all centrifuge tubes on a vortexer using a 50 mL tube adapter. Shake at maximum speed for 0h 2m 0s.

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.

Prepare vacuum filtration units.

Note

One pre-filter/filter unit will be used for each waste sample centrifuge tube, so for one replicate of waste (two waste sample centrifuge tubes), prepare two pre-filter/filter units. One pre-filter/filter unit will be used for each negative control sample, so for one negative control replicate (two centrifuge tubes), prepare one pre-filter/filter units.

8.1.

Place a 2 um pre-filter in a 0.45 um vacuum filtration cup. Repeat for all pre-filter/filter units needed.

Note

Cytiva Whatman Glass Microfiber Prefilters are oriented in the box as they should be placed in the filtration cup. Ensure that the pre-filter is flush with the surface of the filtration cup, and also centered such that sample liquid cannot seep past the pre-filter's edges. Tweezers are useful for proper placement.

Perform filtration for the waste sample.

Note

Waste sample tubes are filtered separately because they easily clog the pre-filter/filter units. Filtrate is combined into one bottle prior to concentration in order to increase the concentration of viral particles in the concentrated eluate.

Centrifuge tubes contain a total of 80 mL of sample (40 mL x 2 tubes). Each tube is filtered with a new filter and pre-filter, and the total volume is collected into one collection bottle.

9.1.

Attach one pre-filter/filter unit to a clean 100 mL pyrex bottle. Decant one waste sample tube into the filtration cup. Begin vacuum filtration by capping the vacuum filtration top and opening the vacuum line.

Citation

The filtrate volume will vary depending on the amount of solid material in the sample. Filtration may occur slowly such filtrate drips at a low yet consistent rate.

9.2.

When filtration is complete, turn off the vacuum line and replace the used pre-filter/filter unit with a fresh one by attaching it to the same pyrex bottle with the waste sample filtrate. Decant the second waste sample tube into the fresh filtration cup. Begin vacuum filtration by capping the vacuum filtration top and opening the vacuum line. When complete, cap the pyrex bottle and set aside.

Note

A filtration cup and pre-filter which are saturated in waste may smell unpleasant. To mitigate this, seal the filtration cup in a bag prior to discarding into a larger biowaste bin.

10.

Perform filtration for the negative control sample.

10.1.

Attach one pre-filter/filter unit to a clean 100 mL pyrex bottle. Decant both centrifuge tubes into the filtration cup. Begin vacuum filtration by capping the vacuum filtration top and opening the vacuum line. When complete, cap the pyrex bottle and set aside.

Part 2: Concentration via InnovaPrep Concentrating Pipette Select

11.

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".

12.

Run the concentration protocol for the filtered waste sample.

12.1.

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.

12.2.

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.

12.3.

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

12.4.

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

Citation

The eluate should be roughly 400 uL. The Zymo DNA/RNA shield reagent is added at a 1:1 ratio of sample:reagent.

13.

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

Note

Make sure to use a fresh Tip for each sample.

14.

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

15.

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.

Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit

16.

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.

17.

Add 1600µL 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.

18.

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.

19.

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

20.

Repeat the previous step.

21.

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

22.

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

23.

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

24.

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.

25.

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.

Concentration and nucleic acid extraction of viruses from aggregated airplane waste

Abstract

Before start

Steps

Reagent Preparation

Part 1: Waste Handling, Dissociation, Filtration

Part 2: Concentration via InnovaPrep Concentrating Pipette Select

Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit

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