Jan 21, 2022

Public workspaceManual Nanotrap Concentration and RNA Extraction for SARS-CoV-2 Viral Capture

DOI
https://dx.doi.org/10.17504/protocols.io.b2uzqex6
Manual Nanotrap Concentration and RNA Extraction for SARS-CoV-2 Viral Capture
  • Matthew Cavallo1,
  • stephen.patrick.hilton 1,
  • jvantas 1,
  • Julia Raymond1,
  • marlene.wolfe 1,
  • Pengbo Liu1,
  • Christine Moe1,
  • Julia Raymond1
  • 1Center for Global Safe WASH, Rollins School of Public Health, Emory University, Atlanta GA USA
Stephen P Hilton
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DOI: https://dx.doi.org/10.17504/protocols.io.b2uzqex6
Protocol Citation: Matthew Cavallo, stephen.patrick.hilton , jvantas , Julia Raymond, marlene.wolfe , Pengbo Liu, Christine Moe, Julia Raymond 2022. Manual Nanotrap Concentration and RNA Extraction for SARS-CoV-2 Viral Capture. protocols.io https://dx.doi.org/10.17504/protocols.io.b2uzqex6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 14, 2021
Last Modified: January 21, 2022
Protocol Integer ID: 55929
Keywords: Nanotrap, manual Nanotrap, wastewater, SARS-CoV-2, COVID-19, magnetic, magnetic virus particles, Ceres, nano, rna extraction for sar, manual nanotrap concentration, rna extraction, wastewater sample, virus, extraction
Funders Acknowledgements:
Ceres Nanosciences – NIH RADx Tech
Grant ID: 75N92021C00012
Abstract
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.
Image Attribution
Fisher Scientific
Materials
Equipment
  • pH probe
  • Magnetic rack (Dynamag)
  • Centrifuge
  • QIAamp Viral RNA Mini Kit (Cat. No. 52904 or 52906)
  • Conical tube (50mL)
  • Eppendorf Research Plus Single Channel Pipette
  • LabGard Biological Safety Cabinet - Class 2 A2 Biosafety Cabinet
  • Autoclave - Amsco Lab 240 Steam Sterilizer

Materials (per sample)
  • 10 μL of BRSV
  • 400 μL of ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
  • 1mL of ReagentMolecular Grade WaterATCCCatalog #60-2450
  • Three 1.7mL tubes
  • 40 μL of Reagent1X PBS (Phosphate-buffered saline )
  • 560 μL of ReagentBuffer AVLQiagenCatalog #19073
  • 5.6 μL of ReagentCarrier RNAThermo FisherCatalog #4382878
  • 560 μL of 96% ethanol
  • 500 μL of ReagentBuffer AW1QiagenCatalog #19081
  • 500 μL of ReagentBuffer AW2QiagenCatalog #19072
  • 60 μL of ReagentBuffer AVEQiagenCatalog #1020953




Protocol materials
ReagentCarrier RNAThermo FisherCatalog #4382878
ReagentBuffer AW1QiagenCatalog #19081
ReagentBuffer AW2QiagenCatalog #19072
ReagentBuffer AVEQiagenCatalog #1020953
ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
ReagentMolecular Grade WaterATCCCatalog #60-2450
Reagent1X PBS (Phosphate-buffered saline )
ReagentBuffer AVLQiagenCatalog #19073
ReagentEthanolP212121Catalog #BE-BDH1156
Reagentdouble distilled water (ddH2O)
Concentration Procedure
Place a 50 mL conical tube in a tube rack.
Vigorously shake the sample to re-suspend solids. Immediately move on to Step 3.
Add Amount50 mL of wastewater to the conical tube inside the biosafety cabinet.

Transfer Amount40 mL of the top supernatant into a new conical tube.

Add Amount10 µL of BRSV to the conical tube.

Add Amount400 µL of ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202 to the sample and invert 2-3 times to mix together and to create a 10:1 sample volume to particle ratio.

Invert samples 3-4 times every 5 minutes at TemperatureRoom temperature for Duration00:20:00 .

20m
Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow the sample to sit in the rack for at least Duration00:10:00 .
Samples must sit in a magnetic rack for at least 10 minutes to allow for particle separation.

10m
Use a pipette to discard the supernatant. Be careful not to disturb the red pellet of Nanotrap particles.

Add Amount1 mL of ReagentMolecular Grade WaterATCCCatalog #60-2450 to the conical tube. Vortex the tube to re-suspend the pellet.

Transfer liquid to a 1.7mL tube. Place tubes on magnetic rack and allow to sit for Duration00:02:00 .

Sample in 1.7 mL tube sits in a magnetic rack for 2 minutes.
A red pellet of Nanotrap particles congregates to the magnetic side of the 1.7 mL tube.
2m
Remove and discard the supernatant. Do not disturb the pellet.
Add Amount140 µL of 1X PBS to the particle pellet.
Note
See "Appendix I: Preparing Buffer Solution (1X PBS)" for instructions on how to prepare 1X PBS.


In a separate 1.7 mL tube, add Amount560 µL of ReagentBuffer AVLQiagenCatalog #19073 and Amount5.6 µL of ReagentCarrier RNAThermo FisherCatalog #4382878 . Mix well by pipetting up and down several times.

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.
Incubate the sample at TemperatureRoom temperature for Duration00:10:00 .

10m
Use a magnetic rack to separate the magnetic Nanotrap particles from the sample. Allow sample to sit on the rack for Duration00:02:00 .

2m
Transfer the supernatant to a 1.7 mL tube and discard the pellet.
Extraction Step (Qiagen Viral RNA Minikit)
11m
Add Amount560 µL of 96% ReagentEthanolP212121Catalog #BE-BDH1156 to sample. Mix well by pipetting up and down several times.

Add Amount630 µL of the sample mix onto a QIAamp Mini column.

Centrifuge sample at full speed for Duration00:01:00 . Once complete, discard the filtrate.
1m
Repeat Step 26 until all of the sample has gone through the column.
Transfer the QIAamp Mini Column into a new collection tube, and discard the tube containing the filtrate.
Open the QIAamp Mini Column and add Amount500 µL of ReagentBuffer AW1QiagenCatalog #19081 . Centrifuge the sample at full speed for Duration00:01:00 . Once complete, discard the filtrate.

1m
Open the QIAamp Mini Column and add Amount500 µL of ReagentBuffer AW2QiagenCatalog #19072 . Centrifuge the sample at full speed for Duration00:03:00 . Once complete, discard the filtrate.

3m
Place the QIAamp Mini Column into a new collection tube and discard the old one containing filtrate. Centrifuge the sample at full speed for Duration00:01:00 to remove any buffer AW2 carryover.

1m
Place the QIAamp Mini Column into a final, labeled tube and discard the old one containing the filtrate
Open the QIAamp Mini Column and add Amount60 µL of ReagentBuffer AVEQiagenCatalog #1020953 and incubate at TemperatureRoom temperature for Duration00:03:00 .

3m
Centrifuge the sample at Centrifigation16000 rpm for Duration00:02:00 .

2m
Separate sample into at least two aliquots. Store in Temperature-80 °C freezer until it is ready for PCR.

Appendix I: Preparing Buffer Solution (1X PBS)
Begin making the 10X PBS Solution by mixing the following:
  • Amount80 g NaCl
  • Amount2 g KCl
  • Amount14.4 g Na2HPO4
  • Amount2.4 g KH2PO4
  • Amount800 mL Ddwater
Adjust the solution to Ph7.4 by adding 5% NaOH and/or 5% HCl.
Note
  • Swirl the mixture after adding either NaOH or HCl.
  • Use a pH probe in between adding NaOH or HCl to see if the pH has reached 7.4 yet.
  • Wash off tip of probe with DI water in between uses.

Add more Reagentdouble distilled water (ddH2O) until the volume is Amount1000 mL .

Dilute the PBS solution by adding Amount100 mL of 10X PBS to Amount900 mL distilled water to create 1X PBS.

Dispense mixture into aliquots and autoclave for Duration00:20:00 . Store at room temperature.