Cellex qCoV Protocol v2

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. 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. 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 25uLReagent 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. 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. 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.