40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations
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Abstract
A one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.
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SciScore for 10.1101/2021.01.26.428302: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources SARS-CoV-2 S protein mutation HV69-70del is also targeted in both reactions to differentiate VOC-202012/01 from the other N501Y containing variants (Table 1). SARS-CoV-2 S protein mutation HV69-70delsuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the …SciScore for 10.1101/2021.01.26.428302: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Experimental Models: Organisms/Strains Sentences Resources SARS-CoV-2 S protein mutation HV69-70del is also targeted in both reactions to differentiate VOC-202012/01 from the other N501Y containing variants (Table 1). SARS-CoV-2 S protein mutation HV69-70delsuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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