Production and use of encapsidated RNA mimics as positive control reagents for SARS-CoV-2 RT-qPCR diagnostics

Peyret, Hadrien; Groppelli, Elisabetta; Clark, David; Eckersley, Nicholas; Planche, Tim; Ma, Julian; Lomonossoff, George P.

Abstract

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.

Citation

Peyret, H., Groppelli, E., Clark, D., Eckersley, N., Planche, T., Ma, J., & Lomonossoff, G. P. (2022). Production and use of encapsidated RNA mimics as positive control reagents for SARS-CoV-2 RT-qPCR diagnostics. Journal of Virological Methods, 300, Article 114372. https://doi.org/10.1016/j.jviromet.2021.114372

• Journal Article Type: Article
• Acceptance Date: Nov 19, 2021
• Online Publication Date: Nov 24, 2021
• Publication Date: 2022-02
• Deposit Date: Mar 5, 2025
• Publicly Available Date: Mar 5, 2025
• Journal: Journal of Virological Methods
• Print ISSN: 0166-0934
• Electronic ISSN: 1879-0984
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 300
• Article Number: 114372
• DOI: https://doi.org/10.1016/j.jviromet.2021.114372
• Keywords: Virus-like particles, Cowpea mosaic virus, SARS-CoV-2, RT-qPCR, Multiplexing, Diagnosis, In-tube control, Pseudogenome
• Public URL: https://nottingham-repository.worktribe.com/output/34350687
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0166093421003116?via%3Dihub