Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2
BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient.
METHOD: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients.
RESULT: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively.
CONCLUSION: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2020 |
|---|---|
| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:17 |
|---|---|
| Enthalten in: |
Virology journal - 17(2020), 1 vom: 28. Dez., Seite 197 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Zhang, Yang [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
COVID-19 |
|---|
| Anmerkungen: |
Date Completed 20.01.2021 Date Revised 20.01.2021 published: Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1186/s12985-020-01467-y |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM319360938 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM319360938 | ||
| 003 | DE-627 | ||
| 005 | 20231225171302.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2020 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1186/s12985-020-01467-y |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1064.xml |
| 035 | |a (DE-627)NLM319360938 | ||
| 035 | |a (NLM)33371898 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Zhang, Yang |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2 |
| 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 20.01.2021 | ||
| 500 | |a Date Revised 20.01.2021 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient | ||
| 520 | |a METHOD: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients | ||
| 520 | |a RESULT: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively | ||
| 520 | |a CONCLUSION: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Validation Study | |
| 650 | 4 | |a COVID-19 | |
| 650 | 4 | |a Highly sensitive | |
| 650 | 4 | |a OSN-qRT-PCR | |
| 650 | 4 | |a SARS-CoV-2 | |
| 650 | 4 | |a ddPCR | |
| 650 | 4 | |a qRT-PCR | |
| 700 | 1 | |a Dai, Chunyang |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Huiyan |e verfasserin |4 aut | |
| 700 | 1 | |a Gao, Yong |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Tuantuan |e verfasserin |4 aut | |
| 700 | 1 | |a Fang, Yan |e verfasserin |4 aut | |
| 700 | 1 | |a Shen, Zuojun |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Lichang |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Zhaowu |e verfasserin |4 aut | |
| 700 | 1 | |a Ma, Xuejun |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Ming |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Virology journal |d 2004 |g 17(2020), 1 vom: 28. Dez., Seite 197 |w (DE-627)NLM151772509 |x 1743-422X |7 nnns |
| 773 | 1 | 8 | |g volume:17 |g year:2020 |g number:1 |g day:28 |g month:12 |g pages:197 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1186/s12985-020-01467-y |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 17 |j 2020 |e 1 |b 28 |c 12 |h 197 | ||