RT Journal Article T1 OmniSARS2: a highly sensitive and specific RT-qPCR-based COVID-19 diagnostic method designed to withstand SARS-CoV-2 lineage evolution A1 Carvalho Correia, Eduarda A1 Calçada, Carla A1 Branca, Fernando A1 Estevez Gomez, Nuria A1 De Chiara Prada, Loretta A1 Varela Rouco, Nair A1 Gallego Garcia, Maria Del Pilar A1 Posada González, David A1 Sousa, Hugo A1 Sousa, João A1 Veiga, Maria Isabel A1 Osório, Nuno S. K1 2420.08 Virus Respiratorios K1 3202 Epidemiología K1 2412 Inmunología AB 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. PB Biomedicines SN 22279059 YR 2021 FD 2021-09-26 LK http://hdl.handle.net/11093/2951 UL http://hdl.handle.net/11093/2951 LA eng NO Biomedicines, 9(10): 1314 (2021) NO Fundação para a Ciência e a Tecnologia | Ref. UIDB / 50026/2020 DS Investigo RD 08-feb-2025