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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Abstract

Tracing the globally circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) phylogenetic clades by high-throughput sequencing is costly, time-consuming, and labor-intensive. We here propose a rapid, simple, and cost-effective amplification refractory mutation system (ARMS)-based multiplex reverse-transcription polymerase chain reaction (PCR) assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. 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 with 0.2–0.6 µM primer concentration, 56–60°C annealing temperature, and 3–5 ng/µl complementary DNA to validate on 24 COVID-19-positive samples. Targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of primers. This multiplex ARMS-PCR assay is a fast, low-cost alternative and convenient to discriminate the circulating phylogenetic clades of SARS-CoV-2.

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Islam, M. T., Alam, A. R. U., Sakib, N., Hasan, M. S., Chakrovarty, T., Tawyabur, M., … Anwar Hossain, M. (2021). A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades. Journal of Medical Virology, 93(5), 2962–2970. https://doi.org/10.1002/jmv.26818

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