Abstract
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.
【저자키워드】 COVID-19, SARS-CoV-2, Diagnosis, RT-PCR, nanopore sequencing, nsp1., 【초록키워드】 coronavirus disease, severe acute respiratory syndrome coronavirus 2, viruses, whole-genome sequencing, Coronavirus disease 2019, coronavirus, pandemic, Mutation, COVID-19 pandemic, Nanopore, diagnostic, SARS-CoV-2 virus, severe acute respiratory syndrome Coronavirus, RT-PCR, binding site, polymerase chain reaction, sensitivity, specificity, validation, nucleocapsid, nasopharyngeal, SARS-CoV-2 genome, management, Patient, nsp1, envelope, nanopore sequencing, respiratory, real-time RT-PCR, PCR assays, false-negative results, Patient management, Molecular assay, Critical, false-negative, RT-PCR assay, binding, Clinical severity, Open reading frame, nonstructural protein 1, COVID-19 patient, Chain Reaction, diagnostic specificity, acute respiratory syndrome, Frame, 95% CI, acute respiratory syndrome coronavirus, 95% confidence interval, E gene, sequence, specimen, PCR assay, SARS-CoV-2 viruses, probes, primer, RT-PCR assays, Primers, molecular assays, saliva specimens, probe, Affect, Genes, caused, addition, expressed, saliva specimen, single-primer amplification, the SARS-CoV-2 genome, 【제목키워드】 whole-genome sequencing, Nanopore, real-time RT-PCR, identification,