TITLE: Cellex qCoV Protocol
AUTHORS: X James Li, Kwok Min Hui,  Don A Gabriel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cellex’s qCOV assay is a viral enzyme activity assay, which detects the activity of coronaviral protease 3CL (chymotrypsin-like protease) in 15 minutes with sensitivity equivalent to 36 cycles of RT-PCR using reagents that are inexpensive and simple to mass produce. We engineered a thermostable firefly luciferase with a 3CL cleavage sequence (mutant luciferase or mLuc). Presence of 3CL enzyme in the sample leads to inactivation of luciferase activity as indicated by reduced light signal. Since luciferase/luciferin is one of the most efficient light generation systems – as few as 2000 molecules could be detected – and 3CL is extremely active with high reaction velocity, this detection system is highly sensitive for detection of coronavirus. </div><div class = "text-block">Until now detection of acute SARS CoV-2 infection has required either molecular assays or antigen assays to detect viral nucleic acids or antigens.  These assays have not provided an adequate diagnosis solution to COVID-19 pandemic management.  The qCOV assay isa completely different detection method and assay: the detection of viral enzyme activity as a means for simple (without sample preparation), rapid (15 min), and sensitive (equivalent to 36 cycles of RT-PCR) detection of active coronavirus infection. </div><div class = "text-block">The assay is a simple homogeneous assay without sample preparation, involving 1) addition of 25 uL Reagent I to 100 uL sample (e.g., throat swab extract) and incubation for 10-15 minutes to allow 3CL digestion, and 2) addition of 25uL Reagent II to enable luciferase reaction and production of light signal. It can be run at point of care settings or in a laboratory in a high throughput fashion. Thousands of samples can be tested in a day with a simple luminometer or other instruments that have luminscence capabilities which are already widely available in nearly every clinical laboratory. </div><div class = "text-block">Unlike a molecular assay which can detect remnants of dead virus, qCOV assay detects only infected cells as the viral particles themselves do not contain 3CL. Upon infection of a cell, viral genome directs synthesis of its preprotein, which contains 3CL. 3CL in the preprotein self cleaves. Cleaved 3CL monomers form a far more active dimer. 3CL is not assembled into viral particles and is present only in infected cells.</div><div class = "text-block">Since all coronaviruses have an enzyme like 3CL, the qCOV assay detects all coronaviruses. The positive samples need to be confirmed with a more specific molecular assay such as RT-PCR. In a community with 5% prevalence of coronavirus infection, including SARS CoV-2, only 5% positive samples need to be further confirmed with RT-PCR, thereby reducing the need for molecular assay by 95%. The qCOV assay is an inhibition assay which produces strong light signal in all negative samples. The negative predictive value is essentially 100%. The qCOV assay can be used not only for diagnosis of an active infection, but also for disease progression monitoring. For example, 3CL activity in blood samples may indicate infection of endothelial cells, which is believed to cause injury like responses including blood vessel clotting. 3CL activity in urine samples may indicate infection of kidney. At present, it appears there are no other good disease monitoring tests for those who are infected with COVID-19.</div></div>

[STEPS]
?. [Sample Collection]
Prep for Sample Collection: Prefill 0.25 mL of sample buffer to a sample extraction vial.  Place the vial in a test tube rack.
?. [Sample Collection]
Sample Collection: Use a throat swab to collect a throat swab specimen, making sure that epithelial cells are collected from the oropharynx.  Insert swab into the posterior pharynx and tonsillar areas.  Rub swab over both tonsillar pillars and posterior oropharynx at least 5 times and avoid touching the tongue, teeth, and gums.
?. [Sample Collection]
Sample Extraction: Insert the swab into the sample extraction vial.  Rub the swab against the tube wall.  While squeezing the tube wall against the swab, slowly remove the swab from the vial.  The extract should be frozen if not used immediately for testing.
?. [Reaction]
Check the ambient temperature.  If it is below  , turn on the oven and set the temperature to  .
25 °C
30 °C
?. [Reaction]
Bring all reagents to room temperature.
?. [Reaction]
Set up a run:
?. [Reaction]
Use the sample buffer as the negative control. If the sample is prepared in a sample buffer different from that of the qCOV Enzyme Rapid Test, the sample buffer should be used as the negative control.  For example, if the sample in VTM is used, VTM solution should be used as the negative control.
?. [Reaction]
Run two negative controls and one positive control for each batch run.
?. [Reaction]
One of negative controls should be placed at the beginning of the run while the other should be placed at the end of the run.
?. [Reaction]
Add   of sample or control to a well.
100 µl
?. [Reaction]
Add  of Reagent I to each well and transfer the plate to the oven; incubate for  .
25 µl
?. [Signal Measurement]
Add   of Reagent II to each well; insert the plate to the luminometer and complete signal measurement within  .
25 µl
?. [Test Result Interpretation]
Calculate the relative activity (RA) of inhibition as follows:        RA = (NC 1 + NC2) ÷ 2 ÷ Sample         The signal is in relative light units (RLU)
?. [Test Result Interpretation]
Use interpretation guide below to interpret result:  ABC1RA Value&nbsp;Interpretation&nbsp;Recommended Action&nbsp;2≥&nbsp;2.00&nbsp;Positive (Active infection)&nbsp;Confirmation by RT-PCR&nbsp;3＜1.50&nbsp;Negative (No infection)&nbsp;No action&nbsp;*The indeterminate results may be interpreted as positive or negative depending on the sample types and / or prevalence of SARS CoV-2 in the community.
ABC1RA Value&nbsp;Interpretation&nbsp;Recommended Action&nbsp;2≥&nbsp;2.00&nbsp;Positive (Active infection)&nbsp;Confirmation by RT-PCR&nbsp;3＜1.50&nbsp;Negative (No infection)&nbsp;No action&nbsp;