Sep 08, 2020

Public workspaceSPOT1 assay

DOI
dx.doi.org/10.17504/protocols.io.bk25kyg6
  • 1University of Illinois at Urbana-Champaign
  • XPRIZE Rapid Covid Testing
  • SPOT
stlane2
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DOI: dx.doi.org/10.17504/protocols.io.bk25kyg6
Protocol Citation: Guanhua Xun, Huimin Zhao, stlane2 2020. SPOT1 assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bk25kyg6
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 07, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41789
Materials
STEP MATERIALS
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentNon-CRISPR nucleaseCatalog #N/A
ReagentNon-primer oligosCatalog #N/A
ReagentE gene primer mixCatalog #N/A
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
ReagentN gene primer mixCatalog #N/A
ReagentReporter Probe 2Catalog #N/A
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentManganese(II) chloride tetrahydrateSigma AldrichCatalog #M3634
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentReporter probe 1Catalog #N/A
ReagentSaliva sampleCatalog #N/A
ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
Protocol materials
ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
ReagentManganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentReporter probe 1Catalog #N/A
ReagentSaliva sampleCatalog #N/A
ReagentNon-primer oligosCatalog #N/A
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentNon-CRISPR nucleaseCatalog #N/A
ReagentE gene primer mixCatalog #N/A
ReagentN gene primer mixCatalog #N/A
ReagentReporter Probe 2Catalog #N/A
ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
ReagentManganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
ReagentNon-primer oligosCatalog #N/A
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
ReagentN gene primer mixCatalog #N/A
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentNon-CRISPR nucleaseCatalog #N/A
ReagentSaliva sampleCatalog #N/A
ReagentReporter Probe 2Catalog #N/A
ReagentE gene primer mixCatalog #N/A
ReagentReporter probe 1Catalog #N/A
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
Using the first provided microcap, a sample is collected into capillary A, containing QuickExtract DNA Extraction Solution (Lucigen). Insert the capillary into the SPOT1 device and press the "Start" button to run the 5-minute pretreatment.



ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050

Equipment
SPOT1 Device
NAME
Incubating fluorometer
TYPE
University of Illinois
BRAND
N/A
SKU
Temperature95 °C
Duration00:05:00

After pretreatment, remove capillary A from the SPOT1 device and use the second provided microcap to transfer a small volume of pretreated sample to capillary B, which contains the SPOT assay mastermix. Insert capillary B into the SPOT1 device and press the "Start" button to initiate the 35-minute detection reaction.
Equipment
SPOT1 device
NAME
Incubating fluorometer
TYPE
University of Illinois
BRAND
N/A
SKU
Temperature63 °C Duration00:30:00
Temperature98 °C Duration00:05:00

SPOT assay mastermix:
Initial concentration Final concentration Amount (μL)
Upper compartment
WarmStart® Bst 2.0 8000 units/mL 160 units/mL 2
WarmStart® RTx 15,000 units/mL 150 units/mL 1
Isothermal amplification buffer 10X 0.5X8
dNTPs 10 mM 0.7 mM 5.6
MgSO4 100 mm 4 mM 3.2
N gene primer mix 10X 0.25X 2
E gene primer mix 10X 0.25X 2
Saliva samples 5
Non-CRISPR nuclease 5 mg/mL or 55 μM 1.375 uM 2
MnCl2 50 mM 0.5 mM 0.8
Non-primer oligos (total 6 oligos) 100 μM 625 nM 3
Reporter probe 1 100 μM 156.25 nM 0.125
Reporter probe 2 100 μM 312.5 uM 0.25
Nuclease-free water 44.025
Total 80
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
Amount2 µL
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
Amount1 µL
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
Amount8 µL
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
Amount5.6 µL Concentration0.7 millimolar (mM)
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
Amount3.2 µL Concentration4 millimolar (mM)
ReagentN gene primer mixCatalog #N/A

ReagentE gene primer mixCatalog #N/A

ReagentSaliva sampleCatalog #N/A

ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S

ReagentNon-CRISPR nucleaseCatalog #N/A

ReagentManganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
Amount0.8 µL Concentration0.5 millimolar (mM)
ReagentNon-primer oligosCatalog #N/A

ReagentReporter probe 1Catalog #N/A


ReagentReporter Probe 2Catalog #N/A

















Result ("Positive"/"Negative"/"Inconclusive") will be displayed on SPOT1 device LCD screen after completion of detection reaction and the 1-minute cooling period.