Sep 05, 2020
Protocol for capture and concentration of viruses from wastewater samples (up to 50 mL) using Magnetic Nanotrap® particles.
This protocol is a draft, published without a DOI.
- Anurag Patnaik1,
- Ben Lepene1,
- Alex Barclay1
- 1Ceres Nanosciences, inc.
- Coronavirus Method Development Community
- 2019-nCoV Wastewater Epidemiology
Protocol Citation: Anurag Patnaik, Ben Lepene, Alex Barclay 2020. Protocol for capture and concentration of viruses from wastewater samples (up to 50 mL) using Magnetic Nanotrap® particles.. protocols.io https://protocols.io/view/protocol-for-capture-and-concentration-of-viruses-bkauksew
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: In development
We are still developing and optimizing this protocol
Created: August 26, 2020
Last Modified: September 05, 2020
Protocol Integer ID: 41012
Keywords: Nanotrap, wastewater, surveillance, COVID, COVID-19, SARS-CoV-2, Pathogen surveillance, Easy workflow,
Abstract
This protocol provides a method for Magnetic Nanotrap® particle-based capture and concentration of viruses from wastewater samples.
Materials
MATERIALS
QIAamp® Viral RNA Mini QiagenCatalog #52906
Microcentrifuge tubesDenville Scientific Inc.Catalog #C2170
PBS 1x without calcium & magnesiumVWR InternationalCatalog #Cat# 21-040-CVR
MicroAmp™ Optical 96-Well Reaction Plate with Barcode & Optical Adhesive FilmsThermo FisherCatalog #4314320
RT-PCR Grade WaterThermo FisherCatalog #AM9935
Mini Vortex MixerCatalog #M10101001
Nanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
RT-PCR Kit
DynaMag™-2 MagnetCatalog #12321D
Sorvall™ Legend™ Micro 21R MicrocentrifugeThermofisherCatalog #75002447
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Follow required Biosafety level requirements.
Virus capture
Virus capture
35m
35m
Place a 50 mL conical tube on a tube rack.
Add 50 mL of wastewater sample to the conical tube.
Let the wastewater sample sit on the benchtop at Room temperature for 00:10:00 . This will allow large aggregates to settle at the bottom of the tube.
Use a pipet to transfer 40 mL of the top clear supernatant, without disturbing the sediment pellet, into a new conical tube.
Add 600 µL of Nanotrap partircles to the sample, and invert 2-3 times to mix the particles.
Incubate samples with Nanotrap particles atRoom temperature for 00:20:00 .
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.00:05:00
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
Qiagen RNA extraction from the Magnetic Nanotrap® particles
30m
30m
Add 140 µL of 1X PBS to the particle pellet.
Add 560 µL of Qiagen lysis buffer from the QIAamp Viral RNA kit to the sample and resuspend the mix by votexing the tube briefly.
Incubate the sample at Room temperature for 00:10:00 .
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.00:00:30
Collect the supernatant in a 2 mL microcentrifuge tube and discard the pellet.
Add 560 µL of 100 % volume ethanol to samples, briefly vortex to ensure the sample is properly mixed.
Add 630 µL of the sample mix onto QIAamp Mini column and close the cap.
Centrifuge the sample for 6000 x g, Room temperature, 00:01:00 .
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
Add the remaining sample from step 14 onto the QIAamp Mini column and close the cap
Centrifuge the sample for 6000 x g, Room temperature, 00:01:00 .
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
Open the QIAamp Mini column and add 500 µL of Buffer AW 1.
Centrifuge the sample for 6000 x g, Room temperature, 00:01:00 .
Place the QIAamp Mini column into a clean 2 mL collection tube and discard the tube containing the filtrate.
Open the QIAamp Mini column and add 500 µL of Buffer AW 2.
Centrifuge the sample for 20000 x g, Room temperature, 00:03:00 .
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
Centrifuge the sample for 20000 x g, Room temperature, 00:01:00 to remove any Buffer AW2 carryover.
Place the QIAamp Mini column into a clean 2 mL collection tube,and discard the tube containing the filtrate.
QIAamp Mini column and add 60 µL of Buffer AVE and incubate at Room temperature for 00:03:00 .
Centrifuge the sample for 6000 x g, Room temperature, 00:01:00 .
The eluate collected in the collection tube is ready for analysis.
Detection
Detection
Use any SARS-CoV-2 RT-PCR detection kit. Follow manufacturer instructions to set up the RT- PCR.
Note
The sample elution can be used for wastewater survillance using MINion or other sequencing platforms.