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Qinghua Hu
Shenzhen CDC
Corresponding Author
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SARS-CoV-2 is a newly emerged coronavirus that was isolated from human infections in recent months. Since drugs and vaccines of Covid-19 are still being developed, accurate pathogen detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been reliably used for the detection and confirmation of SARS-CoV-2 since the beginning of outbreak, whereas isothermal nucleic acid amplification based point of care automated assays has also been considered as a simpler and rapid alternative. However, since these assays have only been developed and applied for clinical use within a short timeframe, their analytical performance has not been adequately compared to-date. We describe a comparative study between a newly developed cross primer isothermal amplification (CPA) assay (Kit A) and five RT-PCR assays (Kits B to F), using clinical diagnosis as the reference standard to evaluate their sensitivity, specificity, predictive values and accuracy analysis. Clinical samples used (n=52) included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprised of positive (n=26) and cleared cases (n=26) by clinical diagnosis. For the CPA assay (Kit A), the sensitivity, specificity, positive and negative predictive values and accuracy were 100%. Among the five RT-PCR kits, Kits B, C and F had good agreement with clinical diagnosis (Kappa≥0.75), Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all assays were statistically significant (P<0.001). The reproducibility of RT-PCR assays was determined using a positive sample that was verified by all assays, with standard deviations (SD) between 0.35 and 0.87, and coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. To further evaluate the CPA assay (Kit A) compared to Kits B and F, throat swabs from close contacts of cases (n=200) were analyzed. All three kits identified the same positive samples and showed total agreement. This is the first comparative study to evaluate a CPA assay and RT-PCR assays for SARS-CoV-2 detection, which could serve as a reference for clinical laboratories and inform testing protocols amid the rapidly evolving COVID-19 pandemic.
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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Review #1 received
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On 02 Dec, 2020
Editor invited
On 02 Dec, 2020
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On 02 Dec, 2020
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Posted 29 May, 2020
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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
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On 30 May, 2020
Editor invited
On 29 May, 2020
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On 26 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
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
The most recent version of this article is available here.
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
First submitted
On 02 Dec, 2020
Version 1
Posted 29 May, 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
The most recent version of this article is available here.
SARS-CoV-2 is a newly emerged coronavirus that was isolated from human infections in recent months. Since drugs and vaccines of Covid-19 are still being developed, accurate pathogen detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been reliably used for the detection and confirmation of SARS-CoV-2 since the beginning of outbreak, whereas isothermal nucleic acid amplification based point of care automated assays has also been considered as a simpler and rapid alternative. However, since these assays have only been developed and applied for clinical use within a short timeframe, their analytical performance has not been adequately compared to-date. We describe a comparative study between a newly developed cross primer isothermal amplification (CPA) assay (Kit A) and five RT-PCR assays (Kits B to F), using clinical diagnosis as the reference standard to evaluate their sensitivity, specificity, predictive values and accuracy analysis. Clinical samples used (n=52) included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprised of positive (n=26) and cleared cases (n=26) by clinical diagnosis. For the CPA assay (Kit A), the sensitivity, specificity, positive and negative predictive values and accuracy were 100%. Among the five RT-PCR kits, Kits B, C and F had good agreement with clinical diagnosis (Kappa≥0.75), Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all assays were statistically significant (P<0.001). The reproducibility of RT-PCR assays was determined using a positive sample that was verified by all assays, with standard deviations (SD) between 0.35 and 0.87, and coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. To further evaluate the CPA assay (Kit A) compared to Kits B and F, throat swabs from close contacts of cases (n=200) were analyzed. All three kits identified the same positive samples and showed total agreement. This is the first comparative study to evaluate a CPA assay and RT-PCR assays for SARS-CoV-2 detection, which could serve as a reference for clinical laboratories and inform testing protocols amid the rapidly evolving COVID-19 pandemic.
The full text of this article is available to read as a PDF.
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