TITLE: Membrane Filtration for SARS-CoV-2 Viral Capture 
AUTHORS: Caleb Centrell, Jamie  VanTassell, Julia Raymond, Marlene K Wolfe, Pengbo, Christine Moe

[DESCRIPTION]
This protocol describes how to perform membrane filtration vacuum technology with wastewater grab samples to allow for SARS-CoV-2 RNA extraction.

[BEFORE_START]
Biosafety Level 2 (BSL-2) certification is required due to the nature of the target pathogen.

[STEPS]
SECTION: Preparation
14. Prepare and collect all supplies necessary for the number of samples that will be concentrated using membrane filtration.
 

SECTION: Sample Processing Procedure
15. Remove the grab sample from the 4°C refrigerator and place under the sterilized biological hood.
SECTION: Sample Processing Procedure
16. Aseptically pour 300 mL of the grab sample into an autoclaved 500mL beaker that is labeled with a sample identifier using labeling tape.

SECTION: Sample Processing Procedure
17. After 300mL of the grab sample has been measured, aseptically pour the aliquoted sample into an autoclaved centrifuge bottle that is labeled with the sample identifier using labeling tape.
 

SECTION: Sample Processing Procedure
18. Using aseptic technique, secure the centrifuged bottle lid onto the centrifuge bottle containing the sample and secure the lid by turning a quarter of the way past tight.
SECTION: Sample Processing Procedure
19. Balance the sample centrifuge bottles using a benchtop scale that has been sterilized using 10% bleach solution followed by 70% ethanol solution.
 

SECTION: Sample Processing Procedure
20. Centrifuge the sample(s) for 5000 rpm, 5 min .

 

 

SECTION: Sample Processing Procedure
21. Remove the centrifuged sample(s) from the centrifuge and carefully place them back under the biological hood.
 

SECTION: Sample Processing Procedure
27. Use a Eppendorf Research Plus Single Channel Pipette to add 10 µL of BRSV aliquot into the sample.
 


SECTION: Sample Processing Procedure
22. Measure out 0.71 g of MgCl2 using benchtop scale paper and an autoclaved chemical spatula.

SECTION: Sample Processing Procedure
23. Pour the measured MgCl2 into the 500mL beaker that was initially used to measure the sample volume. Put a magnetic stir bar into the beaker and stir the sample on the stirrer for 15 min.

SECTION: Sample Processing Procedure
24. Aseptically pour the centrifuged sample into the identically labeled 500mL beaker that now has the MgCl2 to create a final concentration of 25mM.
 

SECTION: Sample Processing Procedure
25. Place a benchtop calibrated pH probe underneath the biological hood and sterilize the probe, excluding the glass measurement part, and workspace with 10% bleach solution followed by 70% ethanol solution.
SECTION: Sample Processing Procedure
26. Adjust the pH to pH 3.5 +/- 0.1 using 5% HCl and 5% NaOH solutions.
 


SECTION: Sample Processing Procedure
28. Cover the opening of the sample beakers with aluminum foil and secure the foil by either wrapping around the edge of the opening or with autoclave tape.
SECTION: Sample Processing Procedure
29. Gently and carefully swirl the sample to allow for homogeneous mixing of the BRSV throughout the sample beaker.
SECTION: Sample Processing Procedure
30. Place the sample beaker into a 4 °C  refrigerator for 30 min. 

SECTION: Sample Processing Procedure
31. While the sample sits in the refrigerator for 30 minutes, place an autoclaved membrane filtration cup into each of the vacuum manifold openings and secure with a quarter turn to create a seal.
SECTION: Sample Processing Procedure
32. Without disturbing the lower half portion of the membrane filtration cup, remove the top cup portion and place topside down onto the benchtop paper that was placed at the start of the procedure preparation.
SECTION: Sample Processing Procedure
33. Using the sterilized forceps, aseptically place a 0.45µM membrane filter paper onto the opening of the membrane filtration cup that is still attached to the vacuum manifold.
SECTION: Sample Processing Procedure
33.1. When initially setting up the vacuum membrane filtration manifold with filter papers, sterilization of forceps can be done a singular time when all membrane filter cups are autoclaved and placed into the biological hood on the vacuum manifold with no sample(s) present.
SECTION: Sample Processing Procedure
33.2. After all membrane filtration cups have a 0.45µM membrane filter paper placed onto their respective opening, reattach the top of the membrane filtration cups to their base.
SECTION: Sample Processing Procedure
33.3. Re-sterilize the forceps and place into an aseptic position for later use after sample processing.
SECTION: Sample Processing Procedure
35. After the 30-minute refrigeration period is over, pour 150 mL of the sample into a membrane filtration cup that has the membrane filter paper and is sealed tightly on the vacuum manifold.
 


SECTION: Sample Processing Procedure
34. Label the membrane filtration cups with their respective sample identifier.
 

SECTION: Sample Processing Procedure
36. Check the seals of the vacuum hoses and vacuum flask to make sure that the seals are secure to prevent leakage of any sample liquid.
SECTION: Sample Processing Procedure
37. Turn the vacuum vent that is attached to the biological hood on to begin the vacuum process, and subsequently turn the valve on the vacuum manifold for each sample that is being processed.
SECTION: Sample Processing Procedure
38. Check the seal on the vacuum hoses and flask once again after the vacuum process has begun.
SECTION: Sample Processing Procedure
39. Throughout the filtration process, frequently check the flow of sample liquid in the membrane filtration cups until the whole sample volume has filtered through the membrane. After 30-45 minutes, if the sample is draining very slowly and a large amount of sample volume still remains to be filtered, follow sub-steps 39.1 - 39.12.
 

SECTION: Sample Processing Procedure
39.1. Retrieve the sample beaker from the 4°C and place under the biological hood.


SECTION: Sample Processing Procedure
39.2. Retrieve a 25mL graduate pipette and an automatic repeater pipette.
SECTION: Sample Processing Procedure
39.3. Turn the vacuum valve off for the respective sample.
SECTION: Sample Processing Procedure
39.4. Pipette the sample that is remaining within the membrane filtration cup back into the sample beaker and measure the volume amount that is removed.
SECTION: Sample Processing Procedure
39.5. After the sample is removed from the membrane filtration cup, remove the top half of the membrane filtration cup and place top down onto the benchtop paper.
SECTION: Sample Processing Procedure
39.6. Using sterilized forceps, roll the membrane filter paper that contains the filtered sample and place into a labeled DNA LoBind tube for RNA extraction.
 

SECTION: Sample Processing Procedure
39.7. Using an Eppendorf Research Plus Single Channel Pipette, place 400 µL of  from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the labeled sample DNA LoBind tube that contains the first sample membrane filter paper.
 

SECTION: Sample Processing Procedure
39.8. Place the sample DNA LoBind tube into a 4℃ refrigerator while the remaining sample volume is filtered through a secondary membrane filter paper.
SECTION: Sample Processing Procedure
39.9. Re-sterilize the forceps and aseptically place a new 0.45 µM membrane filter paper onto the membrane filtration cup opening and reseal using the top half of the membrane filtration cup.
SECTION: Sample Processing Procedure
39.10. Using the same 25mL graduated pipette, replace the measured sample volume into the cup and turn the vacuum valve back to the open position.
SECTION: Sample Processing Procedure
39.11. Once the remaining sample has filtered through the membrane, remove the top half of the cup, roll the membrane filter paper that contains the filtered sample, and place into the labeled DNA LoBind tube that contains the first membrane.
 

SECTION: Sample Processing Procedure
39.12. Add the remaining 400 µL of to the DNA LoBind tube to create a total volume of 800 µL. 

SECTION: Sample Processing Procedure
40. Once the full sample volume (150 mL +/- 15 mL)  has filtered through the membrane, remove the top half of the membrane filtration cup and place top down onto the benchtop paper.
SECTION: Sample Processing Procedure
41. Using sterilized forceps, roll the membrane filter paper that contains the filtered sample and place into a labeled DNA LoBind tube for RNA extraction.
 

SECTION: Sample Processing Procedure
42. Using an Eppendorf Research Plus Single Channel Pipette, place 800 µL of  from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the labeled sample DNA LoBind tube that contains the membrane filter paper.
SECTION: Sample Processing Procedure
43. Store DNA LoBind tubes with filters and Buffer RLT in a 4℃ refrigerator until all samples are processed and ready for RNA extraction.
SECTION: RNA Extraction
44. Insert DNA LoBind tubes that contain the sample filters into a vortex adapter and vortex for 10 min .

SECTION: Post-Sample Processing Clean-Up Procedure
59. Place all benchtop paper and all disposable materials into the benchtop biohazard bag.
SECTION: Post-Sample Processing Clean-Up Procedure
60. Place the benchtop biohazard bag into the correct biohazard floor trash can for future autoclaving.
SECTION: Post-Sample Processing Clean-Up Procedure
61. Sterilize forceps that were used and place into their respective areas for future use.
SECTION: Post-Sample Processing Clean-Up Procedure
62. Place all contaminated membrane filtration cups, sample beakers, centrifuge bottles, and any other glassware into a sink for cleaning prior to autoclaving.
SECTION: Post-Sample Processing Clean-Up Procedure
63. Using concentrated bleach, adequately wash all glassware, centrifuge bottles, and membrane filtration cups and scrub any solids or residue off.
SECTION: Post-Sample Processing Clean-Up Procedure
64. Using lab-grade soap and hot water, adequately wash all glassware, centrifuge bottles, and membrane filtration cups before placing on a drying rack.
SECTION: Post-Sample Processing Clean-Up Procedure
65. Follow current lab procedures for autoclaving and store autoclaved materials for future membrane filtration processing of samples.
SECTION: Post-Sample Processing Clean-Up Procedure
66. Spray 10% bleach solution throughout the biological hood and wipe all surfaces, manifold, and potentially contaminated surfaces with paper towels.
SECTION: Post-Sample Processing Clean-Up Procedure
67. Spray 70% ethanol solution throughout the biological hood and wipe all surfaces, manifold, and potentially contaminated surfaces with paper towels.
SECTION: Post-Sample Processing Clean-Up Procedure
68. Close the biological hood and turn off all vents and lights.
SECTION: RNA Extraction
45. Centrifuge tube for 3 min at maximal speed.
SECTION: RNA Extraction
46. Pipette the available liquid into a new, labeled tube. You may discard the tube with the filter(s) afterwards.
 

SECTION: RNA Extraction
47. Aliquot an equal volume of 70% molecular ethanol to the sample. Mix well by pipetting the mixture up and down several times. Do not vortex after this step.
 

SECTION: RNA Extraction
48. Transfer 700 µL of the sample mixture into a labeled RNeasy spin column. Centrifuge for 30 s at full speed. Discard filtrate. Repeat until all of the sample is filtered through the spin column.
SECTION: RNA Extraction
49. Aliquot 700 µL of  to the RNeasy spin column. Centrifuge for 30 s at full speed. Discard filtrate.
SECTION: RNA Extraction
50. Aliquot 500 µL of  to the RNeasy spin column. Centrifuge for 30 s at full speed. Discard filtrate.
SECTION: RNA Extraction
51. Aliquot 500 µL of  to the RNeasy spin column. Centrifuge for 2 min at full speed. Discard filtrate.
SECTION: RNA Extraction
52. Transfer spin column to new 2 mL collection tube and centrifuge for 1 min at full speed. Discard collection tube and filtrate.
SECTION: RNA Extraction
53. Place the RNeasy spin column into a labeled 1.7 mL microcentrifuge tube. Add 50 µL of RNA-free water to the center of the spin column. Incubate for approximately 1 min at room temperature. 
 

SECTION: RNA Extraction
54. Centrifuge for 1 min at full speed.
SECTION: RNA Extraction
55. Add another 50 µL of RNA-free water to the center of the spin column. Incubate for approximately 1 min at room temperature. 
SECTION: RNA Extraction
58. Store RNA at -20 °C until it is ready for PCR.
SECTION: RNA Extraction
56. Centrifuge for 1 min at full speed.
SECTION: RNA Extraction
57. Close the cap of the 1.7 mL tube containing the final RNA (you will have a final volume of 100 µL).
SECTION: Sterilize Biological Hood
1. Turn on the biological hood vent and wait for airflow to start circulating.
SECTION: Sterilize Biological Hood
2. Carefully remove the vacuum flask and hoses that are attached to the vacuum vent within the biological hood and dispose of any liquid present in the correct biological hazard trash container.
SECTION: Sterilize Biological Hood
3. Wash and rinse the vacuum hoses using a mixture of hot water and concentrated bleach at a 2:1 ratio.
SECTION: Sterilize Biological Hood
4. Pour concentrated bleach solution into the vacuum flask to cover the entire bottom surface.
SECTION: Sterilize Biological Hood
5. Spray 10% bleach solution inside the biological hood and wipe all surfaces with paper towels.
SECTION: Sterilize Biological Hood
6. After spraying the 10% bleach solution, spray 70% ethanol solution inside the biological hood and wipe all surfaces with paper towels.
SECTION: Sterilize Biological Hood
7. Carefully reattach the vacuum flask and vacuum hoses to the vacuum vent within the biological hood.
SECTION: Sterilize Biological Hood
8. Place two pieces of benchtop surface paper underneath the membrane filtration vacuum apparatus that is inside of the sterilized biological hood.
SECTION: Sterilize Forceps
9. Pour 100% ethanol into a smaller autoclaved beaker to a level that allows for the entire end of forceps to be submerged.
SECTION: Sterilize Forceps
10. Carefully light a alcohol lamp burner.
SECTION: Sterilize Forceps
11. Place the forceps that have been submerged in the 100% ethanol and burn the ethanol off to create a visible flame.
SECTION: Sterilize Forceps
12. Place the now sterilized forceps into the biological hood in a position so that the sterilized end is not touching a physical surface to remain aseptic.
SECTION: Sterilize Forceps
13. When forceps are used or come into contact with any surface or filter paper that is contaminated with a sample, they must be re-sterilized using the method given above.