{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,28]],"date-time":"2025-11-28T17:24:46Z","timestamp":1764350686862,"version":"build-2065373602"},"reference-count":24,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,9,26]],"date-time":"2021-09-26T00:00:00Z","timestamp":1632614400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomedicines"],"abstract":"<jats:p>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\u2013sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.<\/jats:p>","DOI":"10.3390\/biomedicines9101314","type":"journal-article","created":{"date-parts":[[2021,9,27]],"date-time":"2021-09-27T01:59:47Z","timestamp":1632707987000},"page":"1314","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5821-0893","authenticated-orcid":false,"given":"Eduarda","family":"Carvalho-Correia","sequence":"first","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4806-909 Guimar\u00e3es, Portugal"}]},{"given":"Carla","family":"Cal\u00e7ada","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4806-909 Guimar\u00e3es, Portugal"},{"name":"Center for Neuroscience and Cell Biology (CNC), University of Coimbra, 3004-504 Coimbra, Portugal"}]},{"given":"Fernando","family":"Branca","sequence":"additional","affiliation":[{"name":"Hospital de Braga, 4710-243 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7281-3137","authenticated-orcid":false,"given":"Nuria","family":"Est\u00e9vez-G\u00f3mez","sequence":"additional","affiliation":[{"name":"CINBIO, Universidade de Vigo, 36310 Vigo, Spain"},{"name":"Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7700-6417","authenticated-orcid":false,"given":"Loretta","family":"De Chiara","sequence":"additional","affiliation":[{"name":"CINBIO, Universidade de Vigo, 36310 Vigo, Spain"},{"name":"Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"given":"Nair","family":"Varela","sequence":"additional","affiliation":[{"name":"CINBIO, Universidade de Vigo, 36310 Vigo, Spain"},{"name":"Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"given":"Pilar","family":"Gallego-Garc\u00eda","sequence":"additional","affiliation":[{"name":"CINBIO, Universidade de Vigo, 36310 Vigo, Spain"},{"name":"Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1407-3406","authenticated-orcid":false,"given":"David","family":"Posada","sequence":"additional","affiliation":[{"name":"CINBIO, Universidade de Vigo, 36310 Vigo, Spain"},{"name":"Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"},{"name":"Department of Biochemistry, Genetics, and Immunology, Universidade de Vigo, 36310 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5795-2131","authenticated-orcid":false,"given":"Hugo","family":"Sousa","sequence":"additional","affiliation":[{"name":"Virology Service and Molecular Oncology and Viral Pathology Group (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), 4200-072 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3249-0035","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Sousa","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4806-909 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2205-8102","authenticated-orcid":false,"given":"Maria Isabel","family":"Veiga","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4806-909 Guimar\u00e3es, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0949-5399","authenticated-orcid":false,"given":"Nuno S.","family":"Os\u00f3rio","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal"},{"name":"ICVS\/3B\u2019s\u2014PT Government Associate Laboratory, 4806-909 Guimar\u00e3es, 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