TITLE: SPOT2 revised protocol
AUTHORS: Huimin Zhao, stlane2 , Guanhua Xun

[STEPS]
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Prepare a new test kit and collect a patient saliva sample.
?. [SPOT testing protocol]
Perform sample pretreatment for 5 minutes at 95°C.If using water baths: Place P-tube in floating rack and insert rack into sous vide water bath preheated to 95°C.  Incubate for 5 minutes for sample pretreatment.If using SPOT carousel device: Place P-tube into carousel and close lid.  On SPOT software, press the "Run Tests" button on the main menu then the "Pretreatment" button to perform a 95°C incubation for 5 minutes.
95 °C
?. [SPOT testing protocol]
Prepare detection reactions:Pair P-tube with a D-tube (Detailed in substep 4.1).Transfer a small volume of pretreated sample to the D-tube (Detailed in substep 4.2).
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Measure test results:If using water baths: Press "Run Tests" on the main menu then of the SPOT dashboard.  Transfer D-tubes one-by-one to the detection slot on SPOT dashboard then press the "Run" button to quantify test results for each D-tube.  The integrated RFID chip on each D-tube will automatically link test result to the patient information entered previously and will be saved to the internal database.If using SPOT carousel device: After completion of the detection reaction, the SPOT carousel will automatically measure the fluorescent output of each D-tube present in the carousel.  The diagnostic result will be linked to the appropriate patient using the integrated RFID antenna and saved to the internal database.
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On the SPOT software main menu, press the "Prepare Kits" button to begin the testing process.  Insert a P-tube into the SPOT dashboard, enter the patient's name, then press "Create" to link the capillary's RFID chip to the patient.
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Provide the prepared P-tube to the patient along with a microcap for saliva collection.  The saliva sample will be collected with the microcap then dispensed into the P-tube, containing QuickExtract DNA Extraction Solution (Lucigen).
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Obtain an unused D-tube and pair it with a completed P-tube using the SPOT dashboard.On the SPOT dashboard main menu, click the "Pair Tubes" button.Tap the P-tube to the RFID antenna on the SPOT dashboard to populate patient information to the screen.Tap the unused D-tube to the RFID antenna on the SPOT dashboard.  After the second RFID tag number populates, press the "Pair" button on the screen.  Patient information assigned during preparation of a test kit will now be coupled to this D-tube.
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Use the provided 20 µL microcap to transfer a small volume of pretreated saliva from the P-tube to the paired D-tube.  Be sure to dispense the pretreated sample into ONLY the top layer of the D-tube, as shown in the diagram below.  Do not disturb the wax dividing layer during transfer of pretreated sample.D-tube contains the SPOT assay mastermix:  ABCD1       Initial concentration    Final concentration    Amount (μL)  2Upper compartment3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    212  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water          44.02517Total          80
ABCD1       Initial concentration    Final concentration    Amount (μL)  2Upper compartment3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    212  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water          44.02517Total          80
2 µl
1 µl
8 µl
5.6 µl
3.2 µl
0.8 µl
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Run detection reaction:If using water baths: Place D-tube in floating rack and insert rack into sous vide water bath preheated to 63°C and incubate for 30 minutes.  After completion of the 63°C incubation, move the floating rack with D-tubes to sous vide water bath preheated to 95°C and incubate D-tubes for 5 minutesIf using SPOT carousel device: Place D-tubes into carousel and close lid.  On SPOT software, click the "Run Tests" button on the main menu then the "Detection" button to perform a full detection reaction (63°C for 30 minutes followed by a 95C for 5 minutes).
63 °C
95 °C
?. [Preparation of equipment and materials]
Gather the provided P-tubes (pretreatment tubes) and D-tubes (detection tubes).  Refrigerate D-tubes until use.Gather microcaps to be used for saliva collection and sample transfer.Plug in keyboard and power cable then power on the SPOT dashboard system.  If using water baths: Power on both sous vide water baths, setting one to 63°C and the other to 95°C.If using SPOT carousel device:  Plug the SPOT dashboard into the SPOT carousel with the included USB cable.  Power on the SPOT carousel system.