Sep 26, 2020

Public workspacePooled sample testing (VTM/UTM) for SARS-CoV-2 using Magnetic Nanotrap® particles for direct RNA extraction.

This protocol is a draft, published without a DOI.
  • 1Ceres Nanosciences, inc.
  • Coronavirus Method Development Community
  • Nanotrap Applications
Anurag Patnaik
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Protocol Citation: Anurag Patnaik, Ben Lepene, Robert Barclay 2020. Pooled sample testing (VTM/UTM) for SARS-CoV-2 using Magnetic Nanotrap® particles for direct RNA extraction.. protocols.io https://protocols.io/view/pooled-sample-testing-vtm-utm-for-sars-cov-2-using-bkyqkxvw
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: September 08, 2020
Last Modified: September 26, 2020
Protocol Integer ID: 41712
Keywords: pooled testing, sample pooling, COVID pooled testing, SARS-CoV-2, Respiratory viruses, Nanotrap, Direct RNA extraction, Magnetic particles, Virus capture, virus concentration, virus detection, viral RNA extraction,
Abstract
This protocol provides a method for detection of SARS-CoV-2 from pooled Viral transport media and Universal transport media samples using Magnetic Nanotrap® particles.
Materials
MATERIALS
ReagentMicrocentrifuge Tubes
ReagentTriton(R) X-100 100mlPromegaCatalog #H5142
ReagentPBS 1x without calcium & magnesiumVWR InternationalCatalog #Cat# 21-040-CVR
ReagentMicroAmp™ Optical 96-Well Reaction Plate with Barcode & Optical Adhesive FilmsThermo FisherCatalog #4314320
ReagentRT-PCR Grade WaterThermo FisherCatalog #AM9935
ReagentMini Vortex MixerCatalog #M10101001
ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
ReagentViral Transport Media (VTM)
ReagentUniversal Transport Media (UTM)
ReagentRT-PCR Kit
ReagentDynaMag™-2 MagnetCatalog #12321D
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Follow required Biosafety level requirements.
Sample preparation
Sample preparation
20m
20m
Pool 8 clinical VTM / UTM samples. Combine Amount500 µL from each sample into a Amount5 mL or Amount15 mL tube.

Pipetting
Add Amount300 µL of Magnetic Nanotrap® particles to the sample.

Pipetting
Incubate samples with Magnetic Nanotrap® particles at TemperatureRoom temperature for Duration00:10:00

Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.Duration00:01:00

Discard the supernatant carefully without disturbing the pellet.
Add Amount500 µL of 1X PBS to the pellet and resuspend to wash.

Wash
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.Duration00:01:00

Discard the supernatant carefully without disturbing the pellet. If required - use a smaller pipette to remove any residual PBS.
Critical
Resuspend particle pellet in Amount50 µL of extraction buffer (Quick vortex if required).



Pipetting
Transfer the resuspension mix to a Amount0.5 mL OR Amount1.5 mL microcentrifuge tube.

Note
Do not heat samples in a Amount5 mL or larger tube. The heat distribution required for lysis is not adequate in larger tube sizes.


Pipetting
Critical
Heat samples at Temperature95 °C for Duration00:05:00

Note
This step can be performed on a heat block or thermocycler.

Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.Duration00:01:00

Collect the supernatant. The sample is ready for analysis.
RT-PCR detection
RT-PCR detection
Use any SARS-CoV-2 RT-PCR detection kit. Follow manufacturer instructions to set up the RT- PCR
PCR