TITLE: Protocol for capture and concentration of viruses from wastewater samples (up to 50 mL)   using Magnetic Nanotrap® particles.
AUTHORS: Anurag Patnaik, Ben Lepene, Alex Barclay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides a method for Magnetic Nanotrap® particle-based capture and concentration of viruses from wastewater samples.</div></div>

[STEPS]
?. [Virus capture]
Place a 50 mL conical tube on a tube rack.
?. [Virus capture]
Add   of wastewater sample to the conical tube.
50 mL
?. [Virus capture]
Let the wastewater sample sit on the benchtop at   for  . This will allow large aggregates to settle at the bottom of the tube.
0 Room temperature
?. [Virus capture]
Use a pipet to transfer  of the top clear supernatant, without disturbing the sediment pellet, into a new conical tube.
40 mL
?. [Virus capture]
Add  of Nanotrap partircles to the sample, and invert 2-3 times to mix the particles.
600 µl
?. [Virus capture]
Incubate samples with Nanotrap particles at for  .
0 Room temperature
?. [Virus capture]
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.
?. Discard the supernatant carefully without disturbing the pellet. If required use a small pipet to remove all the supernatant from the bottom of the tube.
?. [Virus capture]
Discard the supernatant carefully without disturbing the pellet. If required use a small pipet to remove all the supernatant from the tube.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Add  of 1X PBS to the particle pellet.
140 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Add  of Qiagen lysis buffer from the QIAamp Viral RNA kit to the sample and resuspend the mix by votexing the tube briefly.
560 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Incubate the sample at  for  .
0 Room temperature
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Collect the supernatant in a  microcentrifuge tube and discard the pellet.
2 mL
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Add   of  ethanol to samples, briefly vortex to ensure the sample is properly mixed.
560 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Add  of the sample mix onto QIAamp Mini column and close the cap.
630 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for  .
Centrifuge: 6000 34, 1 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Add the remaining sample from step 14 onto the QIAamp Mini column and close the cap
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for  .
Centrifuge: 6000 34, 1 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Open the QIAamp Mini column and add  of Buffer AW 1.
500 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for  .
Centrifuge: 6000 34, 1 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Place the QIAamp Mini column into a clean 2 mL collection tube and discard the tube containing the filtrate.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Open the QIAamp Mini column and add  of Buffer AW 2.
500 µl
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for  .
Centrifuge: 20000 34, 3 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for   to remove any Buffer AW2 carryover.
Centrifuge: 20000 34, 1 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
QIAamp Mini column and add  of Buffer AVE and incubate at  for  .
60 µl
0 Room temperature
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
Centrifuge the sample for  .
Centrifuge: 6000 34, 1 min
?. [Qiagen RNA extraction from the Magnetic Nanotrap® particles]
The eluate collected in the collection tube is ready for analysis.
?. [Detection]
Use any SARS-CoV-2 RT-PCR detection kit. Follow manufacturer instructions to set up the RT- PCR.
The sample elution can be used for wastewater survillance using MINion or other sequencing platforms.