{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T03:54:42Z","timestamp":1780890882358,"version":"3.54.1"},"reference-count":26,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2022,11,8]],"date-time":"2022-11-08T00:00:00Z","timestamp":1667865600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2022,11,8]],"date-time":"2022-11-08T00:00:00Z","timestamp":1667865600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Medical Virology"],"published-print":{"date-parts":[[2023,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world, presenting a new threat to global public human health. Due to the large number of mutations accumulated by SARS\u2010CoV\u20102 Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse\u2010transcription polymerase chain reaction (RT\u2010qPCR), the gold standard diagnostic test for diagnosing coronavirus disease 2019 (COVID\u201019). Thus, we aimed to assess the impact of the currently endemic Omicron sublineages BA.4 and BA.5 on the integrity and sensitivity of RT\u2010qPCR assays used for coronavirus disease 2019 (COVID\u201019) diagnosis via in silico analysis. We employed whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers\/probes and the Omicron VoC viral genome.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>In silico sensitivity of 12 RT\u2010qPCR tests (containing 30 primers and probe sets) developed for detection of SARS\u2010CoV\u20102 reported by the World Health Organization (WHO) or available in the literature, was assessed for specifically detecting SARS\u2010CoV\u20102 Omicron BA.4 and BA.5 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. Mismatches between amplicon regions of SARS\u2010CoV\u20102 Omicron VoC and primers and probe sets were evaluated, and clustering analysis of corresponding amplicon sequences was carried out.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>From the 1164 representative SARS\u2010CoV\u20102 Omicron VoC BA.4 sublineage genomes analyzed, a substitution in the first five nucleotides (C to T) of the amplicon's 3\u2032\u2010end was observed in all samples resulting in 0% sensitivity for assays HKUnivRdRp\/Hel (mismatch in reverse primer) and CoremCharite N (mismatch in both forward and reverse primers). Due to a mismatch in the forward primer's 5\u2032\u2010end (3\u2010nucleotide substitution, GGG to AAC), the sensitivity of the\u00a0ChinaCDC N assay was at 0.69%. The 10 nucleotide mismatches in the reverse primer resulted in 0.09% sensitivity for Omicron sublineage BA.4 for Thai N assay. Of the 1926 BA.5 sublineage genomes, HKUnivRdRp\/Hel assay also had 0% sensitivity. A sensitivity of 3.06% was observed for the\u00a0ChinaCDC N assay because of a mismatch in the forward primer's 5\u2032\u2010end (3\u2010nucleotide substitution, GGG to AAC). Similarly, due to the 10 nucleotide mismatches in the reverse primer, the Thai N assay's sensitivity was low at 0.21% for sublineage BA.5. Further, eight\u00a0assays for BA.4 sublineage retained high sensitivity (more than 97%) and 9 assays for BA.5 sublineage retained more than 99% sensitivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>We observed four assays (HKUnivRdRp\/Hel,\u00a0ChinaCDC N, Thai N, CoremCharite N) that could potentially result in false negative results for SARS\u2010CoV\u20102 Omicron VoCs BA.4 and BA.5 sublineages. Interestingly, CoremCharite N had 0% sensitivity for Omicron Voc BA.4 but 99.53% sensitivity for BA.5. In addition, 66.67% of the assays for BA.4 sublineage and 75% of the assays for BA.5 sublineage retained high sensitivity. Further, amplicon clustering and additional substitution analysis along with sensitivity analysis could be used for the modification and development of RT\u2010qPCR assays for detecting SARS\u2010CoV\u20102 Omicron VoC sublineages.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/jmv.28241","type":"journal-article","created":{"date-parts":[[2022,10,20]],"date-time":"2022-10-20T02:32:09Z","timestamp":1666233129000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["In silico evaluation of the impact of Omicron variant of concern sublineage BA.4 and BA.5 on the sensitivity of RT\u2010qPCR assays for SARS\u2010CoV\u20102 detection using whole genome sequencing"],"prefix":"10.1002","volume":"95","author":[{"given":"Divya","family":"Sharma","sequence":"first","affiliation":[{"name":"Protein Bioinformatics Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences Indian Institute of Technology Madras Chennai Tamil Nadu India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6055-0886","authenticated-orcid":false,"given":"Kin Israel","family":"Notarte","sequence":"additional","affiliation":[{"name":"Department of Pathology Johns Hopkins University School of Medicine Baltimore Maryland USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rey Arturo","family":"Fernandez","sequence":"additional","affiliation":[{"name":"Ateneo de Manila University Professional Schools Rockwell Center Makati Philippines"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Lippi","sequence":"additional","affiliation":[{"name":"Section of Clinical Biochemistry University of Verona Verona Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. Michael","family":"Gromiha","sequence":"additional","affiliation":[{"name":"Protein Bioinformatics Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences Indian Institute of Technology Madras Chennai Tamil Nadu India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8047-338X","authenticated-orcid":false,"given":"Brandon M.","family":"Henry","sequence":"additional","affiliation":[{"name":"Clinical Laboratory, Division of Nephrology and Hypertension Cincinnati Children's Hospital Medical Center Cincinnati Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,11,8]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1515\/dx-2021-0149"},{"key":"e_1_2_11_3_1","unstructured":"Classification of Omicron (B.1.1.529): SARS\u2010CoV\u20102 Variant of Concern.\u00a0Accessed December 30 \u00a02021.https:\/\/www.who.int\/news\/item\/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2008-3"},{"key":"e_1_2_11_5_1","unstructured":"World Health Organization. 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