Sep 09, 2020

Public workspaceXPRIZE SHINE - In-tube Fluorescent SARS-CoV-2 NP Test

DOI
dx.doi.org/10.17504/protocols.io.bk4hkyt6
  • Jon Arizti-Sanz1,2,
  • Catherine A. Freije1,
  • Chloe K. Boehm1,
  • Sameed M. Siddiqui1,3,
  • Allen M. Goodman1,
  • Tinna-Solveig F. Kosoko-Thoroddsen1,
  • A'Doriann Y. Bradley1,
  • Jeremy Johnson1,
  • Pardis C. Sabeti1,4,5,6,7,
  • Cameron Myhrvold1,8,7
  • 1Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA.;
  • 2Harvard-MIT Program in Health Sciences and Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA.;
  • 3Computational and Systems Biology PhD Program, MIT, Cambridge, MA 02139, USA.;
  • 4Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA.;
  • 5Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA.;
  • 6Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.;
  • 7Massachusetts Consortium on Pathogen Readiness, Boston, MA, USA.;
  • 8Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA
  • XPRIZE Rapid Covid Testing
Catherine Freije
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DOI: dx.doi.org/10.17504/protocols.io.bk4hkyt6
Protocol Citation: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold 2020. XPRIZE SHINE - In-tube Fluorescent SARS-CoV-2 NP Test. protocols.io https://dx.doi.org/10.17504/protocols.io.bk4hkyt6
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 in our workspace and it is working.
Created: September 08, 2020
Last Modified: September 09, 2020
Protocol Integer ID: 41833
Keywords: CRISPR, SARS-CoV-2, nucleic acid diagnostic
Disclaimer
The protocol and its content is for informational and academic purposes only. It does not constitute legal, medical, clinical, or safety advice, or otherwise. Content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol is not a substitute for independent professional judgment, advice, diagnosis, or treatment. Any action taken or not taken based on the protocol presented here is strictly at your own risk. You agree that none of the authors, contributors, administrators, nor anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol.
Abstract
This protocol describes how to perform a SHINE in-tube fluorescent assay to detect SARS-CoV-2 RNA from a self-collected nasopharyngeal sample. This protocol is intended for point-of-care use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol requires a transilluminator or another equivalent blue light emitting device. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. bioRxiv (2020).
Image Attribution
biorender.com
Materials
MATERIALS
ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633
ReagentCustom nasopharyngeal swab and collection tube
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)
ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)
STEP MATERIALS
ReagentCustom nasopharyngeal swab and collection tube
ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)
ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)
The necessary volume of FastAmp Viral and Cell Solution is provided in the tube used for sample collection. All enzymatic components, reagents, and compatible buffers required for SARS-CoV-2 detection are included within Lyophilized Reagent Mix B and are reconstituted with Reagent Mix A (see protocol for details). A transilluminator or equivalent blue light emitting device is needed to visualize the assay results. A smartphone or smart device is necessary for automated interpretation of the SARS-CoV-2 detection results using the HandLens application.
Protocol materials
ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633
ReagentCustom nasopharyngeal swab and collection tube
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)
ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)
ReagentCustom nasopharyngeal swab and collection tube
ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)
ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)
ReagentCustom nasopharyngeal swab and collection tube
ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)
ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)
Safety warnings
Please take care with potentially infectious sample material that does not come into contact with the provided viral lysis solution contained within the NP sample collection tube.
Before start
Download the HandLens application on the user-provided smart device (smartphone, tablet, etc.). Clean workspace with disinfectant prior to starting the protocol.
Sample Collection and Viral Lysis
Sample Collection and Viral Lysis
Open the nasopharyngeal (NP) collection tube and rotate the nasal swab (attached to the NP collection tube cap) 4 times around the inside of each nostril. Return the swab to the collection tube and cap the tube.
Nasopharyngeal collection tube contains necessary volume of FastAmp® Viral and Cell Solution.
ReagentCustom nasopharyngeal swab and collection tube

ReagentFastAmp® Viral and Cell Solution for Covid-19 Testing Solution BCatalog #4633

Mix NP sample and FastAmp® Viral and Cell Solution by vortexing the closed sample collection tube for Duration00:00:05 .
Wait Duration00:05:00 , incubating sample at TemperatureRoom temperature , before proceeding to Step 4.



SARS-CoV-2 Detection
SARS-CoV-2 Detection
Pipette Amount15 µL of Reagent Mix A into a single uncapped well of the 96-well plate containing lyophilized Reagent Mix B. Mix by pipetting up and down gently.
ReagentReagent Mix A (In-tube SARS-CoV-2 resuspension mix)

ReagentLyophilized Reagent Mix B (In-tube SARS-CoV-2 detection mix)

Add Amount5 µL sample-viral lysis mix to Reagent Mix A and B well. Mix by pipetting up and down gently. Recap sample.
Wait Duration01:30:00 , incubating sample at TemperatureRoom temperature , before proceeding to Step 7.

In-tube Fluorescent Readout and Automated Analysis
In-tube Fluorescent Readout and Automated Analysis
Visualize the fluorescence of the sample using a transilluminator or equivalent blue light emitting device.
Equipment
13 x 12 cm mini Transilluminator
NAME
Clare Chemical Research
BRAND
DR22A
SKU

With the user-provided smart device such as a smartphone, open the HandLens application and select in-tube as the test type.
Take a photo of the plate, and select upload. The result of the test will appear on the smart device screen.