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A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades
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  • Md. Tanvir Islam,
  • A. S. M. Alam,
  • Najmuj Sakib,
  • Md. Hasan,
  • Tanay Chakrovarty,
  • Md. Tawyabur ,
  • Ovinu Islam,
  • Hassan Al-emran,
  • Md. Jahid,
  • M. Hossain
Md. Tanvir Islam
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A. S. M. Alam
University of Dhaka
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Najmuj Sakib
Jessore University of Science and Technology
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Tanay Chakrovarty
Jessore University of Science and Technology
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Md. Tawyabur
Jessore University of Science and Technology
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Ovinu Islam
Jashore University of Science & Technology
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Hassan Al-emran
Jashore University of Science and Technology
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Md. Jahid
Jessore University of Science and Technology
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M. Hossain
Jessore University of Science and Technology
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Abstract

Tracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders viral genotyping in low-income countries. Here, we propose a rapid, simple, and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. Our multiplex PCR is designed in a mutually exclusive way to identify V-S and G-GH-GR clade variants separately. The pentaplex assay included all five variants and the quadruplex comprised of the triplex variants alongside either V or S clade mutations that created two separate subsets. The procedure was optimized in the primer concentration (0.2-0.6 µM) and annealing temperature (56-60°C) of PCR using a 3-5 ng/µl cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentrations for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is a sample, cost-effective, and convenient that can successfully discriminate against the circulating phylogenetic clades of SARS-CoV-2.

Peer review status:POSTED

11 Oct 2020Submitted to Transboundary and Emerging Diseases
12 Oct 2020Assigned to Editor
12 Oct 2020Submission Checks Completed