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

J Med Virol. 2020 Nov;92(11):2725-2734. doi: 10.1002/jmv.26140. Epub 2020 Jun 19.


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.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; diagnosis; nanopore sequencing; nsp1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / virology
  • COVID-19 Nucleic Acid Testing
  • Female
  • Humans
  • Male
  • Middle Aged
  • Mutation
  • Nanopore Sequencing / methods*
  • Nasopharynx / virology
  • Open Reading Frames
  • RNA, Viral / genetics
  • RNA-Dependent RNA Polymerase / genetics*
  • SARS-CoV-2 / genetics*
  • Saliva / virology
  • Sensitivity and Specificity
  • Viral Nonstructural Proteins / genetics*
  • Whole Genome Sequencing / methods*


  • RNA, Viral
  • Viral Nonstructural Proteins
  • Nsp1 protein, SARS coronavirus
  • RNA-Dependent RNA Polymerase