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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
Ming Li
The First Affiliated Hospital of University of Science and Technology of China
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Virology Journal.
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 30–60%. 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 LoD (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.
Keywords
COVID-19, SARS-CoV-2, qRT-PCR, OSN-qRT-PCR, ddPCR, highly sensitive
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CURRENT STATUS: Published
Version 1
Posted 18 Sep, 2020
Published
On 28 Dec, 2020
No community comments so far
Editorial decision: Minor revision
On 18 Nov, 2020
Review #3 received
Received 15 Nov, 2020
Review #2 received
Received 11 Nov, 2020
Reviewer #3 agreed
On 01 Nov, 2020
Review #1 received
Received 01 Nov, 2020
Reviewers invited
Invitations sent on 29 Oct, 2020
Reviewer #1 agreed
On 29 Oct, 2020
Reviewer #2 agreed
On 29 Oct, 2020
Editor invited
On 15 Sep, 2020
Editor assigned
On 15 Sep, 2020
Submission checks complete
On 14 Sep, 2020
First submitted
On 11 Sep, 2020
Metrics
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Subject Areas
Laboratory Diagnostics
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This preprint is under consideration at Virology Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
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
Ming Li
The First Affiliated Hospital of University of Science and Technology of China
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 1
Posted 18 Sep, 2020
Published
On 28 Dec, 2020
No community comments so far
Editorial decision: Minor revision
On 18 Nov, 2020
Review #3 received
Received 15 Nov, 2020
Review #2 received
Received 11 Nov, 2020
Reviewer #3 agreed
On 01 Nov, 2020
Review #1 received
Received 01 Nov, 2020
Reviewers invited
Invitations sent on 29 Oct, 2020
Reviewer #1 agreed
On 29 Oct, 2020
Reviewer #2 agreed
On 29 Oct, 2020
Editor invited
On 15 Sep, 2020
Editor assigned
On 15 Sep, 2020
Submission checks complete
On 14 Sep, 2020
First submitted
On 11 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Laboratory Diagnostics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Virology Journal.
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 30–60%. 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 LoD (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.
Figure 1
Figure 2
Figure 3
Figure 4