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

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 transcriptionpolymerase chain reaction (RT-PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence-independent singleprimer 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 Wan-Mui Chan and Jonathan Daniel Ip contribute equally.

Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative

“…Except for the variations found throughout the genome, mutations of SARS-CoV-2 virus were also found at the primer-or probe-binding sites [47], which might lower the sensitivity of the current targeting genes. Furthermore, we speculated that the detection of Nsp1 could avoid false-negative results caused by mutations at the primer-or probe-binding sites [48]. Notably, the deletion of 382 nucleotides in the ORF8 gene enhances the transcription of the downstream N gene [26], which might increase the false-negative detection rate of SARS-CoV-2.…”

Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

“…To date, primers and probes used in different RT-PCR assays mainly target ORF1ab, S, E, N regions of SARS-CoV-2 [1,14,17,[19][20][21][22][23][24]. Coronaviruses have a high frequency of mutations and recombination [25][26][27], which may result in mismatches with the currently used primers and probes.…”

“…For analytical sensitivity evaluation, 10-fold serial dilutions of TNA extracted from the SARS-CoV-2 culture isolate were used. For analytical specificity evaluation, TNA extracted from a clinical specimen positive for HCoV-HKU1 and 17 culture isolates of other human coronaviruses and respiratory viruses were used [19,20,31]. For diagnostic performance evaluation, 213 initial clinical specimens including NPA, NPS, throat swab or saliva collected from 213 hospitalized patients (male: female = 112: 101; median age: 48 years; range: 62 days-103 years) with suspected COVID-19 were included for SARS-CoV-2 detection.…”

Symptoms, epidemiology and diagnosis: A mini-review on coronavirus

“…In addition, RdRp gene and E gene were utilized by two groups (Okamaoto et al, 2020;Son et al, 2020). Another group targeted the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes to design primers for RT-PCR assay (Chan et al, 2020). The primer sets design should be performed by selecting the target for amplification and the region of the targeted sequence needs to be unique, specifically for COVID-19.…”