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.
Highlights
The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by the coronavirus SARS-CoV-2, and reverse transcriptase quantitative PCR (RT-qPCR) has been the main technique used to di agnose infections since the start of the pandemic (Corman et al, 2020)
To meet the requirements of different diagnostic systems, two distinct encapsidated mimics are described here: a side-by-side (SBS) mock-positive reaction control and an in-tube (IT) extraction and reac tion positive control (Fig. 1). Both are cowpea mosaic virus (CPMV) virus-like particle (VLP) that contain a syn thetic 1.2 kb RNA pseudogenome construct designed based on the Journal of Virological Methods 300 (2022) 114372 reference SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2)
This construct includes all of the primer binding sites and amplicons of the seven RT-qPCR diagnostic test protocols that were described as technical guidance on the World Health Organisation (WHO) website on 18th March, 2020
Summary
The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by the coronavirus SARS-CoV-2, and reverse transcriptase quantitative PCR (RT-qPCR) has been the main technique used to di agnose infections since the start of the pandemic (Corman et al, 2020). This involves nucleic acid extraction from a patient sample (usually a nasopharyngeal swab in universal transport medium (UTM) or viral transport medium (VTM)), followed by amplification and detection of the extracted nucleic acid by RT-qPCR. Neither naked RNA nor DNA can be used as a control for the nucleic acid extraction step involved in SARS-CoV-2 infection diagnosis. SeraCare commercialises SARS-CoV-2 positive control reagents (AccuplexTM) which are composed of recombinant alphavirus containing segments of the SARS-CoV-2 genome
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