TITLE: SPOT system protocol
AUTHORS: Guanhua Xun, Huimin Zhao, stlane2 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here we report a rapid </span><span style = ":UNDERLINE;">S</span><span>calable and </span><span style = ":UNDERLINE;">Po</span><span>rtable </span><span style = ":UNDERLINE;">T</span><span>esting (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume low-cost access to areas in need of urgent COVID-19 testing capabilities.</span></div></div>

[STEPS]
?. Using the first provided microcap, collect a saliva sample into capillary 1, containing QuickExtract DNA Extraction Solution (Lucigen).  Insert the capillary into the SPOT device and press the "Start" button to run the 5-minute pretreatment.
95 °C
?. After pretreatment, remove capillary 1 from the SPOT device and use the second provided microcap to transfer a small volume of pretreated sample to capillary 2, which contains the SPOT assay mastermix.  Dispense the pretreated sample into only the top layer of the capillary, as shown in the diagram below.  Disturbing the wax dividing layer during sample transfer may lead to a failed reaction.SPOT assay master mix:  ABCD1       Initial concentration    Final concentration    Amount (μL)  2Upper compartment3  WarmStart® Bst 2.0    8000 units/mL  320 units/mL1.64  WarmStart® RTx    15,000 units/mL  300 units/mL0.85  Isothermal amplification buffer    10X  0.5X46  dNTPs    10 mM  0.7 mM5.6  7  MgSO4    100 mm  4 mM3.2  8  N gene primer mix    10X  0.25X2  9  E gene primer mix    10X  0.25X2  10  Saliva samples            5  11Nuclease-free water15.812Total401314Lower compartment15Isothermal amplification buffer10X1X416PfAgo  5 mg/mL or 55 μM    1.375 uM  217  MnCl2  50 mM    0.5 mM  0.818  gDNAs (total 6 oligos)    100 μM    625 nM  319  Reporter probe 1    100 μM    156.25 nM  0.125  20  Reporter probe 2    100 μM    312.5 uM  0.25  21  Nuclease-free water            29.82522Total          40
ABCD1       Initial concentration    Final concentration    Amount (μL)  2Upper compartment3  WarmStart® Bst 2.0    8000 units/mL  320 units/mL1.64  WarmStart® RTx    15,000 units/mL  300 units/mL0.85  Isothermal amplification buffer    10X  0.5X46  dNTPs    10 mM  0.7 mM5.6  7  MgSO4    100 mm  4 mM3.2  8  N gene primer mix    10X  0.25X2  9  E gene primer mix    10X  0.25X2  10  Saliva samples            5  11Nuclease-free water15.812Total401314Lower compartment15Isothermal amplification buffer10X1X416PfAgo  5 mg/mL or 55 μM    1.375 uM  217  MnCl2  50 mM    0.5 mM  0.818  gDNAs (total 6 oligos)    100 μM    625 nM  319  Reporter probe 1    100 μM    156.25 nM  0.125  20  Reporter probe 2    100 μM    312.5 uM  0.25  21  Nuclease-free water            29.82522Total          40
2 µl
1 µl
8 µl
5.6 µl
3.2 µl
0.8 µl
?. Insert capillary 2 into the SPOT device and press the "Start" button to initiate the 35-minute detection reaction.
63 °C
95 °C
?. Result ("Positive"/"Negative"/"Inconclusive") will be displayed on SPOT device LCD screen after completion of detection reaction and the 1-minute cooling period.