CoV2-ID, a MIQE-compliant sub-20-minute 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19

Abstract

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique fiveplex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either separately with separate fluorophores or jointly using the same fluorophore, thus increasing the test’s reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 minutes. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.

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“…The integrity of new batches of SARS-CoV-2 RT-PCR reagents (i.e., primers, probes, PCR master mix, and DNase/RNase free water) should be confirmed before use (Bustin et al, 2020;Huggett et al, 2020). This can be achieved by analyzing 10 to 12 NTC replicates alongside a positive control for each new lot of RT-PCR reagents.…”

Detecting SARS-CoV-2 Orf3a and E ion channel activity in COVID-19 blood samples

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