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

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.