OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution

Abstract

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Evaluation of SARS-CoV-2 Genome Detection By real-time Pcr Assay Using Pooled Specimens

“…Different SARS-CoV-2 lineages could potentially affect the sensitivity of PCR-based detection methods. Simultaneous detection of more than one fragment of the SARS-CoV-2 genome could overcome the false-negative challenge in both individual and pooled samples tests [ 26 , 27 ]. This method has disadvantages, as well.…”

PMMA Microcapsules for the Inactivation of SARS-CoV-2

“…The RT-qPCR reactions were performed using the OmniSARS2 assay. 25 Briefly, the assay detects three different SARS-CoV-2 genes (ORF1ab, S, and E) and the human RNP gene as an internal control. Reactions were set for a final volume of 30 μL reaction, containing 10 μL of RNA sample and the remaining volume of NZYSupreme One-Step RT-qPCR Probe Master Mix, MB414 (2020 NZYTech, Lda, Lisbon, Portugal) and the oligonucleotides at a final concentration of 333 nM each SARS-CoV-2 primer, 84 nM each SARS-CoV-2 probe, 267 nM internal control primer, and 67 nM internal control probe.…”

“…Viral load in the tested samples was calculated using absolute quantification from the Cq standard curve of each viral probe determined with the commercial standard reference, EDX SARS-CoV-2 Standard (SKU: COV019, BioRad, Hercules, CA). 25 Assays performed in nonfabric substrates, with and without functionalization with PMMA or PMMA-H 2 O 2 , were done at least in triplicate, with more tests being carried out under more relevant conditions, to ensure reproducibility. The percentage of viral elimination was calculated using Cq values and respective viral quantification load obtained from the control samples (nonfunctionalized fabric) minus the viral load obtained in the functionalized fabric.…”

“…A previously developed highly sensitive SARS-CoV-2 ribonucleic acid degradation assay was performed to measure the antiviral activity of the PMMA-H 2 O 2 microcapsules. In this assay, the exposure experiments were completed from a fresh swab clinical sample, before nucleic acid extraction and the synthesis of cDNA, to evaluate the degradation of SARS-CoV-2 deoxyribonucleic acids as detailed elsewhere .…”

“…RNA was then extracted following the NZY Viral RNA isolation kit, MB40701 (2020 NZYTech, Lda, Portugal) according to the manufacturer’s instructions. The RT-qPCR reactions were performed using the OmniSARS2 assay . Briefly, the assay detects three different SARS-CoV-2 genes (ORF1ab, S, and E) and the human RNP gene as an internal control.…”