Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing
© 2020 Wiley Periodicals LLC..
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.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2020 |
|---|---|
| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:92 |
|---|---|
| Enthalten in: |
Journal of medical virology - 92(2020), 11 vom: 01. Nov., Seite 2725-2734 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Chan, Wan-Mui [VerfasserIn] |
|---|
| Links: |
|---|
| Anmerkungen: |
Date Completed 24.12.2020 Date Revised 16.07.2022 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1002/jmv.26140 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM310811066 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM310811066 | ||
| 003 | DE-627 | ||
| 005 | 20231225140913.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2020 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1002/jmv.26140 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1036.xml |
| 035 | |a (DE-627)NLM310811066 | ||
| 035 | |a (NLM)32501535 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Chan, Wan-Mui |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing |
| 264 | 1 | |c 2020 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 24.12.2020 | ||
| 500 | |a Date Revised 16.07.2022 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2020 Wiley Periodicals LLC. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a COVID-19 | |
| 650 | 4 | |a RT-PCR | |
| 650 | 4 | |a SARS-CoV-2 | |
| 650 | 4 | |a diagnosis | |
| 650 | 4 | |a nanopore sequencing | |
| 650 | 4 | |a nsp1 | |
| 650 | 7 | |a RNA, Viral |2 NLM | |
| 650 | 7 | |a Viral Nonstructural Proteins |2 NLM | |
| 650 | 7 | |a Nsp1 protein, SARS coronavirus |2 NLM | |
| 650 | 7 | |a EC 2.7.7.48 |2 NLM | |
| 650 | 7 | |a RNA-Dependent RNA Polymerase |2 NLM | |
| 650 | 7 | |a EC 2.7.7.48 |2 NLM | |
| 700 | 1 | |a Ip, Jonathan Daniel |e verfasserin |4 aut | |
| 700 | 1 | |a Chu, Allen Wing-Ho |e verfasserin |4 aut | |
| 700 | 1 | |a Yip, Cyril Chik-Yan |e verfasserin |4 aut | |
| 700 | 1 | |a Lo, Lap-Sum |e verfasserin |4 aut | |
| 700 | 1 | |a Chan, Kwok-Hung |e verfasserin |4 aut | |
| 700 | 1 | |a Ng, Anthony Chin-Ki |e verfasserin |4 aut | |
| 700 | 1 | |a Poon, Rosana Wing-Shan |e verfasserin |4 aut | |
| 700 | 1 | |a To, Wing-Kin |e verfasserin |4 aut | |
| 700 | 1 | |a Tsang, Owen Tak-Yin |e verfasserin |4 aut | |
| 700 | 1 | |a Leung, Wai-Shing |e verfasserin |4 aut | |
| 700 | 1 | |a Kwan, Mike Yat-Wah |e verfasserin |4 aut | |
| 700 | 1 | |a Chua, Gilbert T |e verfasserin |4 aut | |
| 700 | 1 | |a Chung, Tom Wai-Hin |e verfasserin |4 aut | |
| 700 | 1 | |a Hung, Ivan Fan-Ngai |e verfasserin |4 aut | |
| 700 | 1 | |a Kok, Kin-Hang |e verfasserin |4 aut | |
| 700 | 1 | |a Cheng, Vincent Chi-Chung |e verfasserin |4 aut | |
| 700 | 1 | |a Chan, Jasper Fuk-Woo |e verfasserin |4 aut | |
| 700 | 1 | |a Yuen, Kwok-Yung |e verfasserin |4 aut | |
| 700 | 1 | |a To, Kelvin Kai-Wang |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of medical virology |d 1990 |g 92(2020), 11 vom: 01. Nov., Seite 2725-2734 |w (DE-627)NLM000285676 |x 1096-9071 |7 nnns |
| 773 | 1 | 8 | |g volume:92 |g year:2020 |g number:11 |g day:01 |g month:11 |g pages:2725-2734 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1002/jmv.26140 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 92 |j 2020 |e 11 |b 01 |c 11 |h 2725-2734 | ||