Highlights • Novel control reagents for SARS-CoV-2 RT-qPCR diagnostics based on RNA packaged within virus-like particles. • Specific packaging of SARS-CoV-2 – derived RNA in cowpea mosaic virus particles in vivo in the plant Nicotiana benthamiana . • Two different types of controls (In-Tube and Side-By-Side) produced for different applications. • Suitable as controls for nucleic acid extraction, reverse transcription, and polymerase chain reaction steps. • A single bench-scale preparation yields enough purified VLP controls for tens of millions of test reactions. The current gold standard technique for SARS-CoV-2 diagnostics is hydrolysis probe-based RT-qPCR. Reliable testing requires reliable control reagents to monitor the efficiency of RNA extraction, reverse transcription and PCR amplification. Here we describe a custom RNA packaging system from the plant virus cowpea mosaic virus to produce virus-like particles that encapsidate specifically designed portions of the genome of SARS-CoV-2, the causative agent of COVID-19. These encapsidated mimics are highly stable particles which can be used either to spike patient swab samples for use as an in-tube extraction and reaction positive control in multiplex RT-qPCR, or alone as a side-by-side mock-positive control reagent. The selection of sequences in the packaged pseudogenomes ensures that these mimics are compatible with the most commonly used primer/probe combinations for SARS-CoV-2 diagnostics (including German Berlin Charité Hospital, American CDC, and Chinese CDC protocols). The plant transient expression system used to produce these encapsidated mimics is inherently low-cost, and sufficiently high-yielding that a single laboratory-scale preparation can provide enough positive control reagent for millions of tests.
【저자키워드】 SARS-CoV-2, Multiplexing, Diagnosis, RT-qPCR, virus-like particles, Cowpea mosaic virus, In-tube control, Pseudogenome, 【초록키워드】 COVID-19, Genome, virus, diagnostics, Particle, RNA, RNA extraction, Patient, Control, reverse transcription, novel, in vivo, Berlin, VLP, PCR amplification, Combination, protocols, Efficiency, plant virus, Nicotiana benthamiana, Reactions, Virus-like particle, gold standard, sequence, nucleic acid extraction, Charité, multiplex RT-qPCR, positive control, transient expression, Specific, reagent, MONITOR, hydrolysis, polymerase chain, swab sample, produced, can be used, virus particle, purified, American CDC, Chinese CDC, packaged, 【제목키워드】 Production, diagnostics, RNA, positive control, reagent,