key: cord-1054308-gixev6jk
authors: Ahmed, S. M.; Juvvadi, S. R.; Kalapala, R.; Sreemanthula, J. B.
title: Detection of SARS-CoV-2 N501Y mutation by RT-PCR to identify the UK and the South African strains in the population of South Indian state of Telangana
date: 2021-03-28
journal: nan
DOI: 10.1101/2021.03.27.21254107
sha: cf447114f8a8a0b97fb9297aeb3f3ac68b1fa00f
doc_id: 1054308
cord_uid: gixev6jk

Objective: To detect N501Y mutation of the SARS-CoV-2 spike protein by RT-PCR to distinguish (B.1.1.7) UK and (501Y.V2) South African strains from others in the population of Telangana and to determine its clinical implications. Methods: A primer-probe mix that specifically detects the mutated N501Y strain by real time RT-PCR was designed. 93 samples that were reported positive for COVID-19 by our laboratory in the month of February 2021 were tested using our own primer-probe mix for the presence of N501Y by RT-PCR. The results of RT-PCR were validated by Sanger sequencing in representative samples. Sanger sequencing of other defining spike mutations of B.1.1.7 (del 69-70, del 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H) and 501Y.V2 (K417N, E484K, N501Y and D614G) was also investigated. Findings: Out of 93 COVID-19 positive samples, 12 samples are detected positive for N501Y by RT-PCR. Sanger sequencing of these 12 samples further confirmed the presence of N501Y and other mutations that are characteristic of UK strain (B.1.1.7). The South African strain (501Y.V2) is not detected in any of our samples in this study. But, the E484K mutation that is characteristic of 501Y.V2 is detected in one N501Y negative sample. Conclusion: Strain-specific RT-PCR for N501Y was developed and validated with Sanger sequencing. Such strategy facilitates quick surveillance for more transmissible and more vaccine resistant strains.

Sample collection: Nasopharyngeal/Oropharyngeal Swabs collected in Viral Transport Medium which were sent to our laboratory for the detection of SARS-CoV-2 were tested using ICMR approved RT-PCR kits. Among these, 93 Viral RNA templates that tested positive for SARS-CoV-2 were randomly selected and saved.

Primer design: The oligonucleotides ( Table 1) Rox Reference Dye LSR (ST0290, TAKARA), 1µl forward primer N501Y-1 (10 pmol/µl), 1µl reverse primer N501Y-2 (10 pmol/µl), 1µl VIC probe N501Y-3 (10 pmol/µl) and 6 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 28, 2021. ; https://doi.org/10.1101/2021.03.27.21254107 doi: medRxiv preprint 5 min. The primer pair A-F and A-R amplifies a 482 bp product that covers del 69-70 and del 144 mutations. The primer pair B-F and B-R amplifies a 639 bp product that covers E484K, N501Y, A570D and D614G mutations. The primer pair C-F and C-R amplifies a 456 bp product that covers P681H and T716I mutations. The primer pair D-F and D-R amplifies a 605 bp product that covers S982A and D1118H mutations. The amplified products (5 µl) were electrophoresed in 2% agarose gel and subjected to Sanger sequencing on Applied Biosystems SeqStudio Genetic Analyzer by following manufacturer's instructions.

Real time RT-PCR: The amplification plot type was chosen as (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 28, 2021. ; https://doi.org/10.1101/2021.03.27.21254107 doi: medRxiv preprint counterpart T716 provided for comparison (Figure 8 ). The chromatogram showing A982 was presented, the un-mutated counterpart S982 provided for comparison (Figure 9 ). The chromatogram showing H1118 was presented, the un-mutated counterpart D1118 provided for comparison (Figure 10) . The chromatogram showing K484 that was detected in N501Y negative sample was presented, the counterpart E484 provided for comparison ( Figure 11 ).

The primer N501Y-1 specifically binds to the mutated Y501 strain but not to the wild type Eventually, all of the 12 patients recovered well over a course of 3 weeks. Constant continued surveillance for newer variants of the SARS-Cov-2 and observing its clinical implications All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. including any Vaccine-resistant strains is hence much needed to fight and eventually put an end to this pandemic. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 28, 2021. ; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 28, 2021. ; https://doi.org/10.1101/2021.03.27.21254107 doi: medRxiv preprint

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