A potential challenge for any nucleic-acid based testing methods is the potential of the virus to mutate and an analysis of primer binding sites targeted by RT-qPCR assays has indeed shown that a high percentage are mutated 15. Targeting more than one viral gene is advisable as there are reports of significant false negative rates for SARS-CoV-2 RT-qPCR testing 13, 14. Most SARS-CoV-2 RT-qPCR assays target two viral genes, and positive results are most commonly obtained through the hydrolysis of dual-labelled probes 12.
Although a more recent publication describes a streamlined assay with a detection limit of 15 copies, this is based on experiments performed by spiking total human RNA with in vitro synthesised viral transcripts, and the total workflow time remains at 2 h 11. They are broadly comparable, although their reported sensitivity of 500 viral copies per reaction 10 is significantly lower than generally achievable using this technology, and assays differ significantly in the speed of their overall workflow 9. Typical commercial tests use a one tube combined RT and amplification protocol, are carried out in fairly large volumes and use slow protocols with real time data acquisition that result in typical assay times of 1–1.5 h. PCR based assays have the advantage of simplicity of design, easier multiplexing potential and in most cases, greater sensitivity. Isothermal approaches typically require more development and optimisation but have the advantage of speed and are more readily implemented into point-of-care systems 9. SARS-CoV-2 is currently identified using real-time reverse transcription (RT)-qPCR 4, isothermal methodologies 5, 6 or CRISPR 7, 8. As a result, the development of nucleic acid-based tests using different molecular approaches has been rapid. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 as the causal agent of the COVID-19 disease 1, 2, and the ongoing pandemic has highlighted many of the inadequacies inherent in current diagnostic testing regimens 3. 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. 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). 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. 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 min. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test’s reliability and sensitivity. We have used the MIQE guidelines to develop two versions of a unique five plex 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. Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections.
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