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Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection
Qinghua Hu
Shenzhen CDC
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy.
Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients.
Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility.
Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.
Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)
Keywords
SARS-CoV-2, COVID-19, nucleic acid detection, real-time reverse 64 transcriptase PCR (RT-PCR), cross primer isothermal amplification (CPA)
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CURRENT STATUS: Under Review
Version 2
Posted 04 Jan, 2021
No community comments so far
Review #1 received
Received 17 Dec, 2020
Reviewers invited
Invitations sent on 13 Dec, 2020
Reviewer #1 agreed
On 13 Dec, 2020
Editor assigned
On 02 Dec, 2020
Submission checks complete
On 02 Dec, 2020
Editor invited
On 02 Dec, 2020
No comments provided
Reviewer #2 agreed
On 26 Oct, 2020
Review #1 received
Received 05 Aug, 2020
Reviewers invited
Invitations sent on 24 Jul, 2020
Reviewer #1 agreed
On 24 Jul, 2020
Editor assigned
On 30 May, 2020
Editor invited
On 29 May, 2020
Submission checks complete
On 26 May, 2020
First submitted
On 20 May, 2020
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This preprint is under consideration at Annals of Clinical Microbiology and Antimicrobials. 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
Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection
Qinghua Hu
Shenzhen CDC
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: Under Review
Version 2
Posted 04 Jan, 2021
No community comments so far
Review #1 received
Received 17 Dec, 2020
Reviewers invited
Invitations sent on 13 Dec, 2020
Reviewer #1 agreed
On 13 Dec, 2020
Editor assigned
On 02 Dec, 2020
Submission checks complete
On 02 Dec, 2020
Editor invited
On 02 Dec, 2020
No comments provided
Reviewer #2 agreed
On 26 Oct, 2020
Review #1 received
Received 05 Aug, 2020
Reviewers invited
Invitations sent on 24 Jul, 2020
Reviewer #1 agreed
On 24 Jul, 2020
Editor assigned
On 30 May, 2020
Editor invited
On 29 May, 2020
Submission checks complete
On 26 May, 2020
First submitted
On 20 May, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy.
Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients.
Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility.
Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.
Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)