TITLE: Aegea Biotechnologies rapid PCR SARS-CoV-2 test (high sensitivity & specificity; able to detect different strain types)
AUTHORS: Lyle J. Arnold Ph.D., Stella M. Sung Ph.D.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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).</div><div class = "text-block">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</div></div>

[STEPS]
?. Prepare Reaction Mix10ul 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 Table:Reaction components and final concentrations   AB1  Component
      Final Conc.  2  Nuclease
  Free H2O    &nbsp;  3  TaqPath™
  1-Step RT-qPCR Master Mix (4x)       4  AEGEA Switch-Blocker(FAM)
      0.5 uM  5  AEGEA COVID
  Forward Primer    0.5 uM  6  AEGEA COVID
  Reverse Primer     2 uM  7  AEGEA
  RNaseP Forward Primer     0.5 uM  8  AEGEA
  RNaseP Reverse Primer    0.5 uM  9  AEGEA
  RNaseP Probe Alexa 647)    0.8 uM  10  AEGEA COVID
  L-Strain Probe (VIC)     0.8 uM  11  AEGEA COVID
  S-Strain Probe (NED)     0.8 uM  12  RNAseP    Variable
    13  Template
  (RNA-L/RNA-S)    Variable   
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0.5 µl
0.5 µl
2 µl
0.5 µl
0.5 µl
0.8 µl
0.8 µl
0.8 µl
AB1  Component
      Final Conc.  2  Nuclease
  Free H2O    &nbsp;  3  TaqPath™
  1-Step RT-qPCR Master Mix (4x)       4  AEGEA Switch-Blocker(FAM)
      0.5 uM  5  AEGEA COVID
  Forward Primer    0.5 uM  6  AEGEA COVID
  Reverse Primer     2 uM  7  AEGEA
  RNaseP Forward Primer     0.5 uM  8  AEGEA
  RNaseP Reverse Primer    0.5 uM  9  AEGEA
  RNaseP Probe Alexa 647)    0.8 uM  10  AEGEA COVID
  L-Strain Probe (VIC)     0.8 uM  11  AEGEA COVID
  S-Strain Probe (NED)     0.8 uM  12  RNAseP    Variable
    13  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.
10 µl
5 µl
5 µl
?. The oligo mix also contains reagents for an internal adequacy control which targets the human RNAseP RNA transcript.Table:qPCR Cycling Condition   ABCDE1  Cycle Step     Temperature 0C    Time     Cycles       2  Step 1    25°C    2 min    1x       3  RT Reaction    50°C     15 min    1x       4  Initial Polymerase Activation and DNA Denaturation     95°C     2 min    1x       5  Denaturation     95°C     3 sec    45 X       6  Annealing     65°C     10 sec       7  Annealing     52 0C    10 sec    Detection   8  Extension     58 0C    1 min       9  Melt Curve     95°C     15 sec    1x       10       40°C     15 sec    1x    Detection
  During Ramp From 40 0C to 95 0C  11       95°C     1 sec    1x  
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ABCDE1  Cycle Step     Temperature 0C    Time     Cycles       2  Step 1    25°C    2 min    1x       3  RT Reaction    50°C     15 min    1x       4  Initial Polymerase Activation and DNA Denaturation     95°C     2 min    1x       5  Denaturation     95°C     3 sec    45 X       6  Annealing     65°C     10 sec       7  Annealing     52 0C    10 sec    Detection   8  Extension     58 0C    1 min       9  Melt Curve     95°C     15 sec    1x       10       40°C     15 sec    1x    Detection
  During Ramp From 40 0C to 95 0C  11       95°C     1 sec    1x