40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

Korukluoglu, G.; Kolukirik, M.; Bayrakdar, F.; Ozgumus, G. G.; Altas, A. B.; Cosgun, Y.; Ketre Kolukirik, C. Z.

doi:10.1101/2021.01.26.428302

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bioRxiv · 10.1101/2021.01.26.428302

40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

Korukluoglu, G.·Kolukirik, M.·Bayrakdar, F.·Ozgumus, G. G.·Altas, A. B.·Cosgun, Y.·Ketre Kolukirik, C. Z.

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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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Korukluoglu, G., Kolukirik, M., Bayrakdar, F., Ozgumus, G. G., Altas, A. B., Cosgun, Y., Ketre Kolukirik, C. Z.. 2021-01-26. 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations. https://doi.org/10.1101/2021.01.26.428302

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