Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing.

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Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic. Accurate detection of SARS‐CoV‐2 using molecular assays is critical for patient management and the control of the COVID‐19 pandemic. However, there is an increasing number of SARS‐CoV‐2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence‐independent single‐primer amplification and nanopore whole‐genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5′ end of the SARS‐CoV‐2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID‐19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real‐time RT‐PCR assay. The primers and probes are highly specific for SARS‐CoV‐2. Validation with 101 clinical specimens showed that our nsp1 RT‐PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%‐97.2%), which was similar to those of N and E gene RT‐PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%‐100%). The addition of nsp1 for multitarget detection of SARS‐CoV‐2 can avoid false‐negative results due to mutations at the primers/probes binding sites of currently available RT‐PCR assays.