Sep 08, 2020

Public workspaceAegea Biotechnologies rapid PCR SARS-CoV-2 test (high sensitivity & specificity; able to detect different strain types)

DOI
dx.doi.org/10.17504/protocols.io.bk2dkya6
  • Lyle J. Arnold Ph.D., Stella M. Sung Ph.D.1
  • 1Aegea Biotechnologies, Inc.
  • Coronavirus Method Development Community
  • XPRIZE Rapid Covid Testing
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DOI: dx.doi.org/10.17504/protocols.io.bk2dkya6
External link: http://www.aegeabiotech.com
Protocol Citation: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D. 2020. Aegea Biotechnologies rapid PCR SARS-CoV-2 test (high sensitivity & specificity; able to detect different strain types). protocols.io https://dx.doi.org/10.17504/protocols.io.bk2dkya6
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 07, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41765
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Abstract
This protocol is for the Aegea Biotechnologies rapid PCR-based SARS-CoV-2 test. This assay uses patented "Switch-Blocker" technology as well as taqman probes to test for presence of SARS-CoV-2 and simultaneously orthogonally validate. A single amplification reaction is performed, and "Switch Blocker" is used on the forward strand and taqman is used on the reverse strand. The assay design has high sensitivity & specificity--single nucleotide level. Moreover, it is able to detect the SARS-CoV-2 L strain vs the SARS-CoV-2 S strain. The test can be adapted to point of care (Roche LIAT) as well as for different SARS-CoV-2 strains as the virus mutates. A next generation version of the assay could identify the presence of the SARS-CoV-2 L/S strains vs. influenza A/B. Finally, because of the sensitivity and specificity, the Aegea PCR-based SARS-CoV-2 test should be able to use saliva samples, and it is suitable for pooled testing. This protocol is designed for high throughput PCR (96 or 384 well plate formats).


Keywords: PCR, COVID-19, coronavirus, SARS-CoV-2, high throughput, multiplex, Switch-Blocker, taqman, high sensitivity, high specificity, accurate, pooling, saliva, strain types, L-strain, S-strain, combination SARS-CoV-2 and influenza
Materials
STEP MATERIALS
ReagentRNAseP_FP1Catalog #RNAseP_FP1
ReagentAEGEA 1001B_TaqCatalog #AEGEA 1001B_Taq
ReagentAEGEA 1001RPCatalog #AEGEA 1001RP
ReagentAEGEA_1003AF_TaqCatalog #AEGEA_1003AF_Taq
ReagentRNAseP_RP1Catalog #RNAseP_RP1
ReagentTaqPath™ 1-Step RT-qPCR Master MixCatalog #A15300
ReagentAEGEA 1001B_Switch-BlockerCatalog #AEGEA 1001B
ReagentAEGEA 1002B_TaqCatalog #AEGEA 1002B_Taq
ReagentAEGEA 1001cFPCatalog #AEGEA 1001cFP
ReagentNuclease-free WaterCatalog #AM9937
Reagentgblock L-typeCatalog #gblock L-type
Reagentgblock S-typeCatalog #gblock S-type
Protocol materials
Reagentgblock S-typeCatalog #gblock S-type
ReagentRNAseP_RP1Catalog #RNAseP_RP1
ReagentTaqPath™ 1-Step RT-qPCR Master MixCatalog #A15300
ReagentAEGEA 1001cFPCatalog #AEGEA 1001cFP
ReagentNuclease-free WaterCatalog #AM9937
ReagentAEGEA 1001B_Switch-BlockerCatalog #AEGEA 1001B
ReagentAEGEA 1002B_TaqCatalog #AEGEA 1002B_Taq
ReagentRNAseP_FP1Catalog #RNAseP_FP1
ReagentAEGEA 1001B_TaqCatalog #AEGEA 1001B_Taq
Reagentgblock L-typeCatalog #gblock L-type
ReagentAEGEA 1001RPCatalog #AEGEA 1001RP
ReagentAEGEA_1003AF_TaqCatalog #AEGEA_1003AF_Taq
ReagentAEGEA 1001B_TaqCatalog #AEGEA 1001B_Taq
ReagentAEGEA 1002B_TaqCatalog #AEGEA 1002B_Taq
ReagentAEGEA 1001cFPCatalog #AEGEA 1001cFP
ReagentTaqPath™ 1-Step RT-qPCR Master MixCatalog #A15300
ReagentAEGEA_1003AF_TaqCatalog #AEGEA_1003AF_Taq
ReagentAEGEA 1001RPCatalog #AEGEA 1001RP
ReagentRNAseP_FP1Catalog #RNAseP_FP1
ReagentRNAseP_RP1Catalog #RNAseP_RP1
ReagentAEGEA 1001B_Switch-BlockerCatalog #AEGEA 1001B
Reagentgblock L-typeCatalog #gblock L-type
Reagentgblock S-typeCatalog #gblock S-type
ReagentNuclease-free WaterCatalog #AM9937
Prepare Reaction Mix
10ul of the isolated total RNA will be used in a 20ul reaction with TaqPath™ 1-Step RT-qPCR Master Mix (5ul TaqPath Mastermix and 5ul of an oligo mix containing
a. 0.5uM AEGEASwitch-Blocker (FAM),
b. 0.5uM AEGEA COVID Forward Primer C,
c. 2uM AEGEA COVID Reverse Primer,
d. 0.8uM AEGEA L-Strain Probe (VIC),
e. 0.8uM AEGEA S-Strain Probe (NED),
f. 0.5uM AEGEARNaseP Forward Primer
g. 0.5 uM AEGEA RNaseP Reverse Primer
h .0.8uM AEGEA RNaseP Probe Alexa 647

ReagentTaqPath™ 1-Step RT-qPCR Master MixCatalog #A15300

ReagentAEGEA 1001B_Switch-BlockerCatalog #AEGEA 1001B
Amount0.5 µL
ReagentAEGEA 1001cFPCatalog #AEGEA 1001cFP
Amount0.5 µL
ReagentAEGEA 1001RPCatalog #AEGEA 1001RP
Amount2 µL
ReagentRNAseP_FP1Catalog #RNAseP_FP1
Amount0.5 µL

ReagentRNAseP_RP1Catalog #RNAseP_RP1
Amount0.5 µL

ReagentAEGEA_1003AF_TaqCatalog #AEGEA_1003AF_Taq
Amount0.8 µL
ReagentAEGEA 1001B_TaqCatalog #AEGEA 1001B_Taq
Amount0.8 µL

ReagentAEGEA 1002B_TaqCatalog #AEGEA 1002B_Taq
Amount0.8 µL
Table:Reaction components and final concentrations
Component Final Conc.
Nuclease Free H2O  
TaqPath™ 1-Step RT-qPCR Master Mix (4x)
AEGEA Switch-Blocker(FAM) 0.5 uM
AEGEA COVID Forward Primer 0.5 uM
AEGEA COVID Reverse Primer 2 uM
AEGEA RNaseP Forward Primer 0.5 uM
AEGEA RNaseP Reverse Primer 0.5 uM
AEGEA RNaseP Probe Alexa 647) 0.8 uM
AEGEA COVID L-Strain Probe (VIC) 0.8 uM
AEGEA COVID S-Strain Probe (NED) 0.8 uM
RNAseP Variable
Template (RNA-L/RNA-S) Variable









10ul nuclease free water (NTC) or 10ul (combined) gBlock control L/S (5 uL gBlock control L; 5uL gBlock control S) will be run in parallel as negative and positive controls respectively.

ReagentNuclease-free WaterCatalog #AM9937
Amount10 µL

Reagentgblock L-typeCatalog #gblock L-type
Amount5 µL

Reagentgblock S-typeCatalog #gblock S-type
Amount5 µL

The oligo mix also contains reagents for an internal adequacy control which targets the human RNAseP RNA transcript.

Equipment
QuantStudio 5
NAME
qPCR
TYPE
ThermoFisher
BRAND
A34322
SKU
https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/quantstudio-3-5-real-time-pcr-system.html
LINK


Table:qPCR Cycling Condition
Cycle Step Temperature 0C Time Cycles
Step 1 25°C 2 min 1x
RT Reaction 50°C 15 min 1x
Initial Polymerase Activation and DNA Denaturation 95°C 2 min 1x
Denaturation 95°C 3 sec 45 X
Annealing 65°C 10 sec
Annealing 52 0C 10 sec Detection
Extension 58 0C 1 min
Melt Curve 95°C 15 sec 1x
40°C 15 sec 1x Detection During Ramp From 40 0C to 95 0C
95°C 1 sec 1x