tetano
Editor, Senior Moderator
J Med Virol
. 2020 Jun 5.
doi: 10.1002/jmv.26140. Online ahead of print.
Identification of nsp1 Gene as the Target of SARS-CoV-2 Real-Time RT-PCR Using Nanopore Whole Genome Sequencing
Wan-Mui Chan[SUP] 1 [/SUP], Jonathan Daniel Ip[SUP] 1 [/SUP], Allen Wing-Ho Chu[SUP] 1 [/SUP], Cyril Chik-Yan Yip[SUP] 2 [/SUP], Lap-Sum Lo[SUP] 2 [/SUP], Kwok-**** Chan[SUP] 1 [/SUP], Anthony Chin-Ki Ng[SUP] 1 [/SUP], Rosana Wing-Shan Poon[SUP] 2 [/SUP], Wing-Kin To[SUP] 3 [/SUP], Owen Tak-Yin Tsang[SUP] 4 [/SUP], Wai-Shing Leung[SUP] 4 [/SUP], Mike Yat-Wah Kwan[SUP] 5 [/SUP], Gilbert T Chua[SUP] 6 [/SUP], Tom Wai-Hin Chung[SUP] 2 [/SUP], Ivan Fan-Ngai ****[SUP] 7 [/SUP], Kin-Hang Kok[SUP] 1 [/SUP], Vincent Chi-Chung Cheng[SUP] 2 [/SUP], Jasper Fuk-Woo Chan[SUP] 1 2 [/SUP], Kwok-Yung Yuen[SUP] 1 2 [/SUP], Kelvin Kai-Wang To[SUP] 1 2 [/SUP]
Affiliations
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the 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 N, E, and ORF1a/b genes. Using sequence-independent single-primer amplification (SISPA) and nanopore whole-genome sequencing, we have found that the 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, 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 multi-target 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. This article is protected by copyright. All rights reserved.
Keywords: COVID-19; RT-PCR; SARS-CoV-2; diagnosis; nanopore sequencing; nsp1.
. 2020 Jun 5.
doi: 10.1002/jmv.26140. Online ahead of print.
Identification of nsp1 Gene as the Target of SARS-CoV-2 Real-Time RT-PCR Using Nanopore Whole Genome Sequencing
Wan-Mui Chan[SUP] 1 [/SUP], Jonathan Daniel Ip[SUP] 1 [/SUP], Allen Wing-Ho Chu[SUP] 1 [/SUP], Cyril Chik-Yan Yip[SUP] 2 [/SUP], Lap-Sum Lo[SUP] 2 [/SUP], Kwok-**** Chan[SUP] 1 [/SUP], Anthony Chin-Ki Ng[SUP] 1 [/SUP], Rosana Wing-Shan Poon[SUP] 2 [/SUP], Wing-Kin To[SUP] 3 [/SUP], Owen Tak-Yin Tsang[SUP] 4 [/SUP], Wai-Shing Leung[SUP] 4 [/SUP], Mike Yat-Wah Kwan[SUP] 5 [/SUP], Gilbert T Chua[SUP] 6 [/SUP], Tom Wai-Hin Chung[SUP] 2 [/SUP], Ivan Fan-Ngai ****[SUP] 7 [/SUP], Kin-Hang Kok[SUP] 1 [/SUP], Vincent Chi-Chung Cheng[SUP] 2 [/SUP], Jasper Fuk-Woo Chan[SUP] 1 2 [/SUP], Kwok-Yung Yuen[SUP] 1 2 [/SUP], Kelvin Kai-Wang To[SUP] 1 2 [/SUP]
Affiliations
- PMID: 32501535
- DOI: 10.1002/jmv.26140
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the 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 N, E, and ORF1a/b genes. Using sequence-independent single-primer amplification (SISPA) and nanopore whole-genome sequencing, we have found that the 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, 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 multi-target 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. This article is protected by copyright. All rights reserved.
Keywords: COVID-19; RT-PCR; SARS-CoV-2; diagnosis; nanopore sequencing; nsp1.