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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.
Islam, Mohammad Tanvir; Alam, Asm Rubayet Ul; Sakib, Najmuj; Hasan, Mohammad Shazid; Chakrovarty, Tanay; Tawyabur, Mohammad; Islam, Ovinu Kibria; Al-Emran, Hassan M; Jahid, Mohammad Iqbal Kabir; Anwar Hossain, Mohammad.
  • Islam MT; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Alam ARU; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Sakib N; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Hasan MS; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Chakrovarty T; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Tawyabur M; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Islam OK; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Al-Emran HM; Department of Biomedical Engineering, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Jahid MIK; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
  • Anwar Hossain M; Department of Microbiology, Jashore University of Science and Technology, Jashore, Bangladesh.
J Med Virol ; 93(5): 2962-2970, 2021 05.
Article in English | MEDLINE | ID: covidwho-1206825
Preprint
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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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Full text: Available Collection: International databases Database: MEDLINE Main subject: COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study / Randomized controlled trials Topics: Variants Limits: Humans Language: English Journal: J Med Virol Year: 2021 Document Type: Article Affiliation country: Jmv.26818

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Full text: Available Collection: International databases Database: MEDLINE Main subject: COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study / Randomized controlled trials Topics: Variants Limits: Humans Language: English Journal: J Med Virol Year: 2021 Document Type: Article Affiliation country: Jmv.26818