Sep 08, 2020

Public workspaceFLASH amp

DOI
dx.doi.org/10.17504/protocols.io.bk2hkyb6
  • 1Quantification by Design, Stanford University
  • XPRIZE Rapid Covid Testing
eesha.sharma.phd
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DOI: dx.doi.org/10.17504/protocols.io.bk2hkyb6
Protocol Citation: eesha.sharma.phd 2020. FLASH amp. protocols.io https://dx.doi.org/10.17504/protocols.io.bk2hkyb6
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: September 08, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41769
Keywords: Xprize,
Disclaimer
This method is currently used for research and development, not for diagnostic purposes.
Abstract
FLASH amp is a method that allows for room temperature detection and amplification of a viral RNA sequence of interest submitted as part of the Xprize competition. The main components of the reaction are not included here and are given generic names such as enzyme X as they are currently not protected under IP. The steps outlined with tubes labeled with the generic names would allow any lab to reproduce the method.

Version 1 of this method outlines the protocol for a fluorescence-based readout that can be used with a qPCR machine or TECAN style plate reader.

Version 2 (to be released shortly) will be a colorimetric readout that can be assessed by eye.

The outlined protocol assumes the user performing this in a laboratory setting will setup reactions in a clean room and analyze results in a seperate post-amplification room. In addition, since our amplification is rapid and at room temperature, care but be taken to add ligand to the master mix quickly and the reactions be sealed.
Materials
STEP MATERIALS
ReagentPyrophosphatase, Inorganic (E.coli) - 50 unitsNew England BiolabsCatalog #M0361L
ReagentCorning® Low Volume 384-well Black Flat Bottom Polystyrene Catalog #3821BC
Protocol materials
ReagentPyrophosphatase, Inorganic (E.coli) - 50 unitsNew England BiolabsCatalog #M0361L
ReagentCorning® Low Volume 384-well Black Flat Bottom Polystyrene Catalog #3821BC
ReagentPyrophosphatase, Inorganic (E.coli) - 50 unitsNew England BiolabsCatalog #M0361L
ReagentCorning® Low Volume 384-well Black Flat Bottom Polystyrene Catalog #3821BC
Sample collection
Sample collection
Sample should be collected 1 hour after any food is consumed.
10 minutes before collection rinse mouth well with water.
Collect passive drool or pooled salive below tongue into collection tube.
Set-up
Set-up
Resuspend freeze dried master mix 1 with Amount7.04 µL of water

Resuspend freeze dried master mix 2 in Amount1 µL water and add to above



AddAmount0.3 µL
ReagentPyrophosphatase, Inorganic (E.coli) - 50 unitsNew England BiolabsCatalog #M0361L

Add Amount0.5 µL

Undisclosed enzyme Z

Add Amount0.5 µL
Undisclosed Enzyme Y

Add Amount0.13 µL
Undisclosed Enzyme X


Put mix in
ReagentCorning® Low Volume 384-well Black Flat Bottom Polystyrene Catalog #3821BC
Add Amount0.5 µL of sample and seal plate.


Note
A large master mix of the above components can be mixed and aliquoted into wells.

Data collection
Data collection
Place plate in TECAN plate reader.

Equipment
SPARK
NAME
Microwell plate reader
TYPE
TECAN
BRAND
SPARK
SKU
https://www.tecan.com/blog/spark-multimode-microplate-reader-for-high-performance-cell-based-fluorescence-assays
LINK


Set gain to 120, excitation 480 and emission 550.
A qPCR machine can also be used.
Collect data at T= 30 min.
Data can also be collected every 1s for 30 min to monitor kinetics of reaction in the particular plate reader being used if not TECAN SPARK reader.

Fluorescence values of >4000 are positive and <4000 are negative.

Expected result