A rapid and cost‐effective multiplex ARMS‐PCR method for the simultaneous genotyping of the circulating SARS‐CoV‐2 phylogenetic clades

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 mu...

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Bibliographic Details
Published in:	Journal of medical virology Vol. 93; no. 5; pp. 2962 - 2970
Main Authors:	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
Format:	Journal Article
Language:	English
Published:	United States Wiley Subscription Services, Inc 01-05-2021 John Wiley and Sons Inc
Subjects:	Amplification ARMS Assaying clade Complementary DNA Coronaviridae Coronaviruses Cost-Benefit Analysis COVID-19 COVID-19 - diagnosis COVID-19 - virology COVID-19 Nucleic Acid Testing - economics COVID-19 Nucleic Acid Testing - methods DNA sequencing Genotype Genotyping Humans multiplex PCR Multiplex Polymerase Chain Reaction Multiplexing Mutation mutations Phylogenetics Phylogeny Polymerase chain reaction Reproducibility of Results Respiratory diseases SARS-CoV-2 - classification SARS-CoV-2 - genetics SARS-CoV-2 - isolation & purification SARS‐CoV‐2 Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Virology COVID-19 multiplex PCR ARMS SARS-CoV-2 mutations clade
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Summary:	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. Highlights Multiplex ARMS‐PCR (amplification refractory mutation system‐polymerase chain reaction) method for genotyping major severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2 clades). Identify the mutated region of circulating phylogenetically SARS‐CoV‐2 clades. PCR conditions were optimized and validated to identify V–S and G–GH–GR clade.
Bibliography:	Mohammad Tanvir Islam, ASM Rubayet Ul Alam, and Najmuj Sakib contributed equally to this study. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0146-6615 1096-9071
DOI:	10.1002/jmv.26818