TITLE: Manual Nanotrap Concentration and RNA Extraction for SARS-CoV-2 Viral Capture
AUTHORS: Matthew   Cavallo, Stephen P Hilton, Jamie  VanTassell, Julia  Raymond, Marlene K Wolfe, Pengbo   Liu, Christine  Moe

[DESCRIPTION]
This protocol details a method for SARS-CoV-2 capture and concentration through the use of Nanotrap® Magnetic Virus Particles from 40mL of wastewater sample.

[STEPS]
SECTION: Concentration Procedure
1. Place a 50 mL conical tube in a tube rack.
SECTION: Concentration Procedure
2. Vigorously shake the sample to re-suspend solids. Immediately move on to Step 3.
SECTION: Concentration Procedure
3. Add 50 mL of wastewater to the conical tube inside the biosafety cabinet.

SECTION: Concentration Procedure
4. Transfer 40 mL of the top supernatant into a new conical tube.

SECTION: Concentration Procedure
5. Add 10 µL of BRSV to the conical tube.

SECTION: Concentration Procedure
6. Add 400 µL of  to the sample and invert 2-3 times to mix together and to create a 10:1 sample volume to particle ratio.

SECTION: Concentration Procedure
7. Invert samples 3-4 times every 5 minutes at Room temperature for 20 min.    

SECTION: Concentration Procedure
8. Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow the sample to sit in the rack for at least 10 min.
  

SECTION: Concentration Procedure
9. Use a pipette to discard the supernatant. Be careful not to disturb the red pellet of Nanotrap particles.

SECTION: Concentration Procedure
10. Add 1 mL of   to the conical tube. Vortex the tube to re-suspend the pellet.

SECTION: Concentration Procedure
11. Transfer liquid to a 1.7mL tube. Place tubes on magnetic rack and allow to sit for 2 min. 

     
SECTION: Concentration Procedure
13. Add 140 µL of 1X PBS to the particle pellet.
 


SECTION: Concentration Procedure
14. In a separate 1.7 mL tube, add 560 µL of   and 5.6 µL of  . Mix well by pipetting up and down several times.

SECTION: Concentration Procedure
15. In a regular, non-magnetic rack, add the Buffer AVL and carrier RNA to the particle pellet. Suspend the pellet by using the pipette to wash the sides of the conical tube with the lysis buffer, carrier RNA, and 1X PBS mixture.
SECTION: Concentration Procedure
16. Incubate the sample at Room temperature for 10 min.

SECTION: Concentration Procedure
17. Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow sample to sit on the rack for 2 min.

SECTION: Concentration Procedure
18. Transfer the supernatant to a 1.7 mL tube and discard the pellet.
SECTION: Extraction Step (Qiagen Viral RNA Minikit)
19. Add 560 µL of 96%   to sample. Mix well by pipetting up and down several times.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
20. Add 630 µL of the sample mix onto a QIAamp Mini column. 

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
22. Repeat Step 26 until all of the sample has gone through the column. 
SECTION: Extraction Step (Qiagen Viral RNA Minikit)
24. Open the QIAamp Mini Column and add 500 µL of  . Centrifuge the sample at full speed for 1 min. Once complete, discard the filtrate.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
25. Open the QIAamp Mini Column and add 500 µL of  . Centrifuge the sample at full speed for 3 min. Once complete, discard the filtrate.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
26. Place the QIAamp Mini Column into a new collection tube and discard the old one containing filtrate. Centrifuge the sample at full speed for 1 min to remove any buffer AW2 carryover.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
27. Place the QIAamp Mini Column into a final, labeled tube and discard the old one containing the filtrate
SECTION: Extraction Step (Qiagen Viral RNA Minikit)
28. Open the QIAamp Mini Column and add 60 µL of  and incubate at Room temperature  for 3 min.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
29. Centrifuge the sample at 16000 rpm for 2 min.

SECTION: Extraction Step (Qiagen Viral RNA Minikit)
30. Separate sample into at least two aliquots. Store in -80 °C freezer until it is ready for PCR.

SECTION: Concentration Procedure
12. Remove and discard the supernatant. Do not disturb the pellet.
SECTION: Extraction Step (Qiagen Viral RNA Minikit)
21. Centrifuge sample at full speed for 1 min. Once complete, discard the filtrate.
SECTION: Extraction Step (Qiagen Viral RNA Minikit)
23. Transfer the QIAamp Mini Column into a new collection tube, and discard the tube containing the filtrate.
SECTION: Appendix I: Preparing Buffer Solution (1X PBS)
31. Begin making the 10X PBS Solution by mixing the following:
80 g NaCl 
2 g KCl 
14.4 g  Na2HPO4
2.4 g KH2PO4 
800 mL Ddwater
SECTION: Appendix I: Preparing Buffer Solution (1X PBS)
32. Adjust the solution to pH 7.4 by adding 5% NaOH and/or 5% HCl.
 

SECTION: Appendix I: Preparing Buffer Solution (1X PBS)
33. Add more  until the volume is 1000 mL. 

SECTION: Appendix I: Preparing Buffer Solution (1X PBS)
34. Dilute the PBS solution by adding 100 mL of 10X PBS to 900 mL distilled water to create 1X PBS.

SECTION: Appendix I: Preparing Buffer Solution (1X PBS)
35. Dispense mixture into aliquots and autoclave for 20 min. Store at room temperature.