Sep 08, 2020

Public workspaceSPOT2 protocol

DOI
dx.doi.org/10.17504/protocols.io.bk28kyhw
  • 1University of Illinois at Urbana-Champaign
  • XPRIZE Rapid Covid Testing
stlane2
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DOI: dx.doi.org/10.17504/protocols.io.bk28kyhw
Protocol Citation: Huimin Zhao, stlane2, Guanhua Xun 2020. SPOT2 protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bk28kyhw
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: 41792
Materials
STEP MATERIALS
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentE gene primer mixCatalog #N/A
ReagentNon-primer oligosCatalog #N/A
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentN gene primer mixCatalog #N/A
ReagentSaliva sampleCatalog #N/A
ReagentManganese(II) chloride tetrahydrateSigma AldrichCatalog #M3634
ReagentReporter probe 1Catalog #N/A
ReagentReporter Probe 2Catalog #N/A
ReagentNon-CRISPR nucleaseCatalog #N/A
Protocol materials
ReagentNon-primer oligosCatalog #N/A
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentReporter Probe 2Catalog #N/A
ReagentN gene primer mixCatalog #N/A
ReagentSaliva sampleCatalog #N/A
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentE gene primer mixCatalog #N/A
ReagentManganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
ReagentReporter probe 1Catalog #N/A
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentNon-CRISPR nucleaseCatalog #N/A
ReagentNon-CRISPR nucleaseCatalog #N/A
ReagentReporter Probe 2Catalog #N/A
ReagentWarmStart RTx Reverse Transcriptase - 250 rxnsNew England BiolabsCatalog #M0380L
ReagentIsothermal Amplification Buffer - 6.0 mlNew England BiolabsCatalog #B0537S
ReagentSaliva sampleCatalog #N/A
ReagentNuclease-free Water - 25 mlNew England BiolabsCatalog #B1500S
ReagentManganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
ReagentNon-primer oligosCatalog #N/A
ReagentReporter probe 1Catalog #N/A
ReagentMagnesium Sulfate (MgSO4) Solution - 6.0 mlNew England BiolabsCatalog #B1003S
ReagentE gene primer mixCatalog #N/A
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentN gene primer mixCatalog #N/A
ReagentBst 2.0 WarmStart DNA Polymerase - 8,000 unitsNew England BiolabsCatalog #M0538L
Label test kit bag with patient's name and a unique sample number for the test run. Provide a 20 uL microcap and PCR tube and have patient collect a saliva sample and place it into then seal the PCR tube. Label PCR tube with sample number matching the test kit bag.
Place PCR tubes into floating rack and insert into sous vide water bath at 95C. Incubate for 5 minutes for sample pretreatment.



Equipment
Thermal Immersion Cooker
NAME
Sous Vide - low cost waterbath
TYPE
Wancle
BRAND
P00252
SKU
Temperature95 °C Duration00:05:00

During incubation, scan barcodes on test kits and enter patient identifying information into the SPOT2 device.

Equipment
SPOT2 Device
NAME
Data logging fluorometer
TYPE
University of Illinois
BRAND
N/A
SKU

Equipment
USB Laser Barcode Scanner
NAME
Barcode Scanner
TYPE
WoneNice
BRAND
WN3300
SKU

After completion of pretreatment, use the second provided 20 uL microcap to transfer a small volume of saliva from the PCR tube to the capillary tube, containing SPOT assay mastermix. Place capillary tubes into floating rack and insert into sous vide water bath at 63C for 30 minutes.
Temperature63 °C Duration00:30:00
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


Transfer floating capillary rack to sous vide at 95C for 5 minutes.
Temperature95 °C Duration00:05:00

Individually transfer capillary tubes to SPOT2 device and press "Run" on the SPOT software. An integrated RFID chip on each capillary will automatically link test result to the patient information entered previously.

Equipment
SPOT2 Device
NAME
Data logging fluorometer
TYPE
University of Illinois
BRAND
N/A
SKU