Sep 08, 2020

Public workspaceFluorescent-Reporter Based Assay

DOI
dx.doi.org/10.17504/protocols.io.bk5rky56
  • 1University of Florida
s.rananaware
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DOI: dx.doi.org/10.17504/protocols.io.bk5rky56
Protocol Citation: Piyush Jain, Long T Nguyen, Santosh Rananaware 2020. Fluorescent-Reporter Based Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bk5rky56
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 08, 2020
Protocol Integer ID: 41873
Keywords: CRISPR, SARS-CoV-2, COVID-19 Diagnostic,
Abstract
The CRISPR-Enhance SARS-CoV-2 detection kit has been designed to detect fragments of the Nucleocapsid (“N”) gene and Envelope gene (E) of SARS-CoV-2. An included third target is the human RNase P POP7 gene (“RP”) which serves as a control for the extraction of the clinical sample in the absence of a positive SARS-CoV-2 result. Amplification can be performed using a heat block, and CRISPR complex activation and reporter cleavage can be run in a standard microplate reader capable of fluorescence detection. The entire reaction from RT-LAMP amplification to CRISPR-based detection of the target analytes can be performed in approximately one hour.

The CRISPR-Enhance kit comprises of two steps. Step one is a reverse transcriptase loop-mediated amplification (RT-LAMP) where targeted SARS-CoV-2 genomic RNA is reverse transcribed to DNA, and this DNA is amplified by a strand-displacing DNA polymerase. Step two is the transcription of the amplified DNA to activate the collateral cleavage activity of a CRISPR complex programmed to the target RNA sequence. This collateral activity results in cleavage of nucleic acid reporters, resulting in a fluorescent readout detected by a plate reader.
Guidelines
All procedures should be performed in a BSL2 laboratory, and specimens should be handled within a Biological Safety Cabinet. All necessary safety precautions should be taken according to the Laboratory guidelines. Precautions must also be taken to prevent cross-contamination of samples.
Materials
MATERIALS
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
STEP MATERIALS
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
Protocol materials
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
Safety warnings
  1. Handle all infectious samples with appropriate CDC approved methods
  2. Wear appropriate PPE such as lab coats, gloves, N95 respirators, safety goggles etc when handling infectious samples
  3. Discard all biohazard waste appropriately
  4. Clean all work surfaces with bleach and IPA after use
Nucleic Acid Extraction
Nucleic Acid Extraction
18m
18m

  • The CRISPR-Enhance SARS-CoV-2 detection kit uses QuickExtract™ RNA Extraction Kit
ReagentQuickExtract™ RNA Extraction KitLucigenCatalog #QER090150
  • Add Amount10 µL of patient sample to Amount10 µL of pre-aliquoted QuickExtract solution.
  • Heat the above mixture at Temperature65 °C forDuration00:15:00 followed by Temperature98 °C for Duration00:03:00 .

18m
RT-LAMP Master Mix Preparation
RT-LAMP Master Mix Preparation
  • Label a new Amount1.5 mL microcentrifuge tube for each target ( N, E and RP) and prepare a RT-LAMP Master Mix consisting of the WarmStart®Colorimetric LAMP 2X Master Mix with UDG.
ReagentWarmStart®Colorimetric LAMP 2X Master Mix with UDG (Cat.No. M1804S)New England BiolabsCatalog #M1804S
and the appropriate 10x Primer Mix using the recipe in Table 1 below. Make enough of each master mix for all samples to be tested and the necessary controls for each run.


Reagent NameVolume per reactionTotal Volume
WarmStart®Colorimetric LAMP 2X Master Mix with UDG12.5 µL12.5 µL x (N+1)
10x Primer Mix (N, E or RP)2.5 µL2.5 µL x (N+1)
RNAse-Free Water5 µL5 µL x (N+1)
Total Volume20 µL20 µL x (N+1)
Table 1: Target Specific RT-LAMP Master Mix Recipe
N = number of extracted samples plus number of controls. Prepare enough for 1 extra (N + 1) sample to allow for overage during reaction set-up.
RT-LAMP Amplification
RT-LAMP Amplification
  • Label a strip tube (Amount0.2 mL ) with the target name (e.g. N) and strip number corresponding to each sample.
  • Add Amount20 µL of the RT-LAMP Master Mix from the previous step into one well for each sample and control to be amplified. Repeat for the remaining 2 targets using a new strip for each target (e.g. E or RP)
  • Add Amount5 µL of extracted RNA in each respective strip tube containing the RT-LAMP Master mix. Vortex the strip tube for Duration00:00:03 and spin down forDuration00:00:03 in microcentrifuge with a Amount0.2 mL tube adaptor.

ReagentVolume per reaction
RT-LAMP Master Mix20 μL
RNA Sample or Controls5 μL
Total Volume25 μL
Table 2: RT-LAMP Assay Components and reaction volume
  • Heat the mixture at Temperature65 °C for Duration00:40:00


CRISPR-Cas Reaction Preparation
CRISPR-Cas Reaction Preparation
  • Preheat a fluorescence microplate reader to Temperature37 °C .
  • For each target tested label a Amount1.5 mL tube with the target name (e.g. N, E or RNASE-P) and “Cas Mix”. Prepare a CRISPR Cas Master Mix using the following recipe in Table 3 below, scaling as required for the number of assays to be run (one Cas assay for every RT-LAMP reaction).
  • Incubate the mixture at Temperature37 °C for Duration00:15:00
  • Pulse vortex forDuration00:00:03 and spin down for Duration00:00:03 in a microcentrifuge after all components are added.


Reagent NameVolume per ReactionVolume Total
NEB 2.1 Buffer1.2 μL1.2 μL x (N+1)
3 μM crRNA (N or E or RP)0.8 μL0.8 μL x (N+1)
3 μM lbCas12a0.4 μL0.4 μL x (N+1)
RNAse-Free Water9.6 μL9.6 μL x (N+1)
Total Volume12 μL12 μL x (N+1)
Table 3: Target CRISPR Cas Master Mix Recipe
N = number of extracted samples plus number of controls. Prepare enough for 1 extra (N + 1) sample to allow for overage during reaction set-up.

  • In a separateAmount1.5 mL microcentrifuge tube prepare the fluorecence reporter as per Table 4


Reagent NameVolume per ReactionVolume Total
FAM-FQ0.2 μL0.2 μL x (N*+1)
RNAse-Free Water25.8 μL25.8 μL x (N*+1)
Total Volume12 μL12 μL x (N*+1)
Table 4: Flourescence Reporter Mix Recipe
N* = number of extracted samples multiplied by the total number of genes plus number of controls. Prepare enough for 1 extra (N* + 1) sample to allow for overage during reaction set-up




CRISPR-Cas Detection
CRISPR-Cas Detection
  • Add 26 μL of the Fluorescent reporter Mix made in step 4 to each well of a 384 well-plate corresponding to the number of samples and controls for every gene (N, E or RP).
  • Add 2 μL of the RT-LAMP product from step 3 to each well containing the fluorescent reporter.
  • Add 12 μL of the CRISPR-Cas master mix to the wells containing the corresponding RT-Lamp product and fluorescent reporter.
  • Seal the plate with an Optical seal
  • Open the plate reader software to create a read procedure. Set temperature to 37°C
  • Select "Kinetic" run reading with a total read time of 30 min, and data collection intervals at 2.5 mins
  • Save experiment in a designated place with an appropriate unique name
  • When plate loader extdends, load plate. Ensure plate is loaded in correct orientation. Read the data.