TITLE: Skim Milk Flocculation and RNA Extraction for SARS-CoV-2 Viral Capture
AUTHORS: Jillian  Dunbar, Stephen P Hilton, Jamie  VanTassell, Julia  Raymond, Marlene K Wolfe, Pengbo   Liu, Christine  Moe

[DESCRIPTION]
This protocols describes the use of Skim Milk Flocculation to concentrate and extract SARS-CoV-2 from Moore swab wastewater samples. Included in this protocol are: materials and equipment, steps for processing Moore swabs, and steps for concentrating and extracting the virus using the Qiagen RNeasy Mini Kit.

[STEPS]
SECTION: Preparing Solutions for Procedure
64. Prepare the NaPP stock by dissolving 1 g of NaPP in 100 mL of  . This should be prepared once a month, and should be autoclaved before use. 

SECTION: Day 2 - Process Moore Swab
64. Autoclave 10 mL of NaPP stock, 10 mL of Tween 80 stock, and 1 mL of Antifoam A stock.

64. 
64. 
SECTION: Moore Swab Processing
3. If using the potato ricer method: 
  

SECTION: Moore Swab Processing
7. Pour only the supernatant back into the flask (should be approximately 250 mL).
 

SECTION: Moore Swab Processing
8. Adjust the pH of the sample to pH 3.5 using 5% HCl and 5% NaOH using a pH probe.

SECTION: Moore Swab Processing
9. Aliquot 10 µL of 104 Bovine Respiratory Syncytial Virus (BRSV) as a positive control to the sample. Mix well by pipetting the BRSV up and down 20 times in the sample before expelling it.
 

SECTION: Moore Swab Processing
10. Add 1 % volume of Skim Milk Solution (see Appendix II for preparation steps) to the sample volume.
 

SECTION: RNA Extraction
16. Gently scrape off and discard a large portion of the pellet, leaving behind a thin film of pellet in the centrifuge bottle.

SECTION: RNA Extraction
20. Centrifuge tube for 3 min at full speed.
SECTION: RNA Extraction
23. 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
24. Aliquot 700 µL of  to the RNeasy spin column. Centrifuge for 30 s at full speed. Discard filtrate. 

SECTION: RNA Extraction
25. Aliquot 500 µL of  to the RNeasy spin column. Centrifuge for 30 s at full speed. Discard filtrate. 

SECTION: RNA Extraction
26. Add 500 µL of  to the RNeasy spin column. Centrifuge for 2 min at full speed. Discard filtrate. 

SECTION: RNA Extraction
27. Transfer spin column to new 2 mL collection tube and centrifuge for 1 min at full speed. Discard filtrate and collection tube.
SECTION: RNA Extraction
28. Place the RNeasy spin column into a 1.7 mL labeled microcentrifuge tube. Aliquot 50 µL of  (from RNeasy kit). Incubate for approximately 1 min at room temperature. 
 


SECTION: RNA Extraction
29. Centrifuge for 1 min at full speed.

SECTION: RNA Extraction
30. Aliquot another 50 µL of  into the the RNeasy spin column in the 1.7 mL labeled microcentrifuge tube. Incubate for approximately 1 min at room temperature. 

SECTION: RNA Extraction
33. Store RNA at -20 °C until it is ready for PCR.

SECTION: RNA Extraction
17. Aliquot 800 µLof  from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the centrifuge bottle. 
SECTION: RNA Extraction
15. Pour supernatant out (you will not use this liquid for RNA extraction).
SECTION: RNA Extraction
18. Mix well by pipetting the pellet and   up and down about 30 times or until the mixture looks homogeneous.

SECTION: RNA Extraction
19. Pipette 800 µL of homogenized mixture into a labeled 2 mL DNA LoBind microcentrifuge tube.
SECTION: RNA Extraction
21. Transfer all of the sample into a new labeled tube. 
SECTION: Moore Swab Processing
12. Place sample on shaker   .
 

SECTION: RNA Extraction
14. Centrifuge sample for 30 min at 12000 rpm.
SECTION: RNA Extraction
22. Aliquot 800 µL  of 70% molecular ethanol. 
 

SECTION: Moore Swab Processing
6. Centrifuge for 15 min at 5000 rpm.
 

SECTION: Moore Swab Processing
4. Ensure all of the centrifuge bottles have similar masses (+/- 0.5g) so that the centrifuge is balanced.
 

SECTION: RNA Extraction
13. Pour sample from flask into sterilized and labeled centrifuge bottle.
SECTION: Moore Swab Processing
11. Place aluminum foil over the opening of the flask.

SECTION: Moore Swab Processing
2. If using a Lab Paddler Stomacher:
SECTION: Moore Swab Processing
2.2. Place Moore swab sample in a clean stomacher bag with 100 mL of Elution Solution (see Appendix I for preparation steps).

SECTION: Moore Swab Processing
2.1. Set the stomacher settings to paddle at speed 2 for 30 s .

SECTION: Moore Swab Processing
2.5. Repeat Steps 2.1 - 2.4 until you have 300 mL of sample volume.

SECTION: Moore Swab Processing
2.4. Fold the stomacher bag in half length-wise and pour the fluid into a labeled centrifuge bottle.
 

SECTION: Moore Swab Processing
3.1. Place Moore swab sample in a potato ricer and squeeze all the liquid out into a 600 mL beaker. 
 

SECTION: Moore Swab Processing
3.2. Pour the squeezed liquid into a labeled 500 mL Erlenmeyer flask.
 

SECTION: Moore Swab Processing
3.3. Place the Moore swab back in the beaker. Add 100 mL of Elution Solution (see Appendix I for preparation steps) into the beaker.
SECTION: Moore Swab Processing
3.4. Gently knead the Moore swab 10 times using the pointed end of a clean 50 mL conical tube. Then, use the top/flat surface of the tube to knead the swab 10 more times.
 

SECTION: Moore Swab Processing
3.5. Turn the swab over and repeat Step 3.4.
SECTION: Moore Swab Processing
3.6. Put the swab back into the potato ricer, and squeeze all the liquid out into the beaker.
SECTION: Moore Swab Processing
3.7. Pour the liquid into the 500 mL Erlenmeyer flask.
SECTION: Moore Swab Processing
3.8. Repeat Steps 3.3 through 3.7 until there is 300 mL of sample liquid in the 500 mL Erlenmeyer flask.


SECTION: Moore Swab Processing
3.9. Pour contents into a labeled centrifuge bottle.
SECTION: Moore Swab Processing
5. Wipe down the benchtop scale after use using 10% bleach followed by 70% ethanol.
SECTION: Moore Swab Processing
1. Two alternative methods are provided herein for initial swab processing: Lab Paddler Stomacher (Step 2) or a Potato Ricer (Step 3).
SECTION: Appendix I - Prepare Elution Solution
34. Prepare the Tween 80 stock by dissolving 1 mL of   in 100 mL of distilled water.

SECTION: Appendix I - Prepare Elution Solution
35. Prepare the Antifoam A stock by dissolving 1 mL of Antifoam A in 100 mL of distilled water. 

SECTION: Appendix I - Prepare Elution Solution
36. Prepare the NaPP stock by dissolving 1 g of NaPP in 100 mL of distilled water.

SECTION: Appendix I - Prepare Elution Solution
37. Prepare the 10X PBS Solution
SECTION: Appendix I - Prepare Elution Solution
37.1. Mix together:
80 gNaCl 
2 gKCl 
14.4 g  Na2HPO4
2.4 g KH2PO4  
800 mL Ddwater
SECTION: Appendix I - Prepare Elution Solution
37.2. Adjust the solution to pH 7.4  by adding 5% NaOH and/or 5% HCl.
 


SECTION: Appendix I - Prepare Elution Solution
37.3. Add more  until the volume is 1000 mL .

SECTION: Appendix I - Prepare Elution Solution
38. Create the Elution Solution by mixing the following:
 880 mL of distilled water
100 mL  of 10 X PBS Solution
 10 mL  of NaPP stock
 10 mL  of Tween 80 stock
 1 mL of Antifoam A stock.

SECTION: Appendix I - Prepare Elution Solution
39. Autoclave Elution Solution before using.
SECTION: Appendix II - Prepare Skim Milk Solution
40. Dissolve 5 g of   in 100 mL  of distilled water using a magnetic stir bar.
SECTION: Appendix II - Prepare Skim Milk Solution
41. Autoclave Skim Milk Solution before using.
SECTION: Moore Swab Processing
2.3. Place bag in stomacher so that the top of the bag is above the stomacher and close the handle. The stomacher will begin to paddle the swab for the programed time.
SECTION: RNA Extraction
32. Close the cap of the 1.7 mL tube containing the final RNA (you will have a final volume of 100 µL).

SECTION: RNA Extraction
31. Centrifuge for 1 min at full speed.