CoV2-ID, a MIQE-compliant sub-20-min 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 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. 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. Viral targets can be detected either individually 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 min. 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.

RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

“…The performance of assay G, which uses the original mismatched RdRP_SARSr-P1 probe, was compared to assay H, which substitutes that probe with the corrected ALT-P1dg sequence. The performance of both was compared to that of a published SARS-CoV-2 assay (CoV2-ID) [39]. Three replicate assays using the PCRBio 1-step RT-qPCR master mix recorded equivalent Cqs, with G:H ∆Cqs of 0.42±0.71, 1.01±0.50 and -0.05±0.45, respectively (Figure 6A).…”

Sample Adequacy Control (SAC) Lowers False Negatives and Increases the Quality of Screening: Introduction of “Non-Competitive” SAC for qPCR Assays

“…and (d) the control of sample processing/extraction. These types of quality controls should not be added externally [31] but should be intrinsically present in the sample [1,3,16,21,22,32]. SAC unifies these quality assurance controls.…”

“…Quality control requirements for screening assays: The MIQE rules are among some of the primary quality requirements to be followed when validating a new screening qPCR assay [ 21 , 22 , 23 , 24 ]. These rules also stipulate that quantitative, or semi-quantitative qPCR analysis can be performed by relative quantification with a second biomarker control, equivalent to SAC [ 22 , 25 ].…”

“…For example, even though emergency uses of different COVID-19 tests are authorized, recommendations made by CDC ( or , accessed on 6 June 2021) and WHO ( , accessed on 6 June 2021) which assure quality of testing must include: (a) control of sampling, (b) control of reaction inhibition, (c) control of the chemical integrity of biomarker (RNA, or DNA, or protein…) and (d) the control of sample processing/extraction. These types of quality controls should not be added externally [ 31 ] but should be intrinsically present in the sample [ 1 , 3 , 16 , 21 , 22 , 32 ]. SAC unifies these quality assurance controls.…”

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus — Worldwide, 2021

“…One of the most critical mutations was the D614G of the spike protein gene (S), which is a replacement of aspartic acid (D) with glycine (G). The transmission of S-G614 mutants was stronger than that of S-D614 mutants because a newly-formed serine protease called elastase-2 in the S-G614 mutant could lead to an increase in enzyme cleavage efficiency and infectivity (3)(4).…”