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However, there is an increasing number of SARS\u2010CoV\u20102 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real\u2010time reverse transcription\u2010polymerase chain reaction (RT\u2010PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence\u2010independent single\u2010primer amplification and nanopore whole\u2010genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5\u2032 end of the SARS\u2010CoV\u20102 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID\u201019 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real\u2010time RT\u2010PCR assay. The primers and probes are highly specific for SARS\u2010CoV\u20102. Validation with 101 clinical specimens showed that our nsp1 RT\u2010PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%\u201097.2%), which was similar to those of N and E gene RT\u2010PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%\u2010100%). The addition of nsp1 for multitarget detection of SARS\u2010CoV\u20102 can avoid false\u2010negative results due to mutations at the primers\/probes binding sites of currently available RT\u2010PCR assays.<\/jats:p>","DOI":"10.1002\/jmv.26140","type":"journal-article","created":{"date-parts":[[2020,6,5]],"date-time":"2020-06-05T13:44:24Z","timestamp":1591364664000},"page":"2725-2734","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":35,"title":["Identification of nsp1 gene as the target of SARS\u2010CoV\u20102 real\u2010time RT\u2010PCR using nanopore whole\u2010genome sequencing"],"prefix":"10.1002","volume":"92","author":[{"given":"Wan\u2010Mui","family":"Chan","sequence":"first","affiliation":[{"name":"State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, Queen Mary Hospital The University of Hong Kong  Hong Kong 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