TITLE: Concentration and nucleic acid extraction of viruses from aggregated airplane waste
AUTHORS: Ari N Machtinger, Olivia S Hershey, William J Bradshaw, Daniel P Rice, Michael R McLaren

[DESCRIPTION]
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.

[GUIDELINES]
RNA processing and handling: Please review Protocol Note: Working with RNA Samples before handling RNA samples.

[STEPS]
SECTION: Reagent Preparation
1. 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.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
2. 
 
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.
SECTION: Part 1: Waste Handling, Dissociation, Filtration
3. 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 20 mL of waste into two 50 mL centrifuge tubes each. Repeat for desired number of sample replicates. For a negative control, add 20 mL of 1X PBS to two centrifuge tubes each using a fresh 50 mL serological pipette, for a total volume of 40 mL.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
5. Cap and parafilm all bottles and centrifuge tubes.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
6. Place all centrifuge tubes on a vortexer using a 50 mL tube adapter. Shake at maximum speed for 2 min.

SECTION: Part 1: Waste Handling, Dissociation, Filtration
7. Transfer all centrifuge tubes from the vortexer to a sonication bath, and sonicate for 1 min   at 40 kHz. Use a paper towel to dry the tubes when finished.
SECTION: Part 1: Waste Handling, Dissociation, Filtration
8. Prepare vacuum filtration units.
 

SECTION: 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".
SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
12. Run the concentration protocol for the filtered waste sample.
SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
12.1. Remove the maintenance tip and place a fresh Ultra CPT into the tip port. Lower the tip into the sample.
 

SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
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.
 

SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
12.3. While holding a 5 mL centrifuge tube under the Tip, press "Elute" and catch the foam that is dispensed.
SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
12.4. After the foam degasses, add 400 µL of the Zymo DNA/RNA shield reagent.
 

SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
13. Run the concentration protocol for the negative control. Perform as is done for the waste sample.
 

SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
14. Incubate samples with added Zymo DNA/RNA shield reagent at room temperature for 30 min. If samples are sitting for longer then store at 4 °C

SECTION: Part 2: Concentration via InnovaPrep Concentrating Pipette Select
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.
SECTION: 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.
 

SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
17. Add 1600 µL of Viral DNA/RNA buffer to the sample (a 2:1 ratio) and vortex briefly to mix.
 

SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
18. 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, 2 min. Discard the flow-through in the miniprep waste container. 

Repeat until full lysate volume is processed.
SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
19. Add 500 µL of Viral Wash Buffer to the column, centrifuge for 12.000 x g, 30 s and discard the flow-through.
SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
20. Repeat the previous step.
SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
21. Add 500 µL ethanol (95-100%) to the column and centrifuge for 12000 x g, 1 min to ensure complete removal of the wash buffer.

SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
22. Transfer the column to a clean collection tube, and centrifuge at 12000 x g, 1 min to remove any remaining ETOH.
 

SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
23. Carefully, transfer the column into a 1.5 mL nuclease-free tube.
SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
24. Add 50 µL DNase/RNase-Free Water directly to the column matrix and incubate at RT for 1 min. Centrifuge for 12000 x g, 30 s to collect the eluate.
SECTION: Part 3: Nucleic Acid Extraction - Zymo quick-DNA/RNA Viral Kit
25. Place all extracted nucleic acids in a freezer set to -80 °C
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
8.1. Place a 2 um pre-filter in a 0.45 um vacuum filtration cup. Repeat for all pre-filter/filter units needed.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
9. Perform filtration for the waste sample.
 


 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
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.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
10. Perform filtration for the negative control sample.
SECTION: Part 1: Waste Handling, Dissociation, Filtration
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.
 

SECTION: Part 1: Waste Handling, Dissociation, Filtration
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.
SECTION: Part 1: Waste Handling, Dissociation, Filtration
4. Add 18 mL 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.