A rapid and cost‐effective multiplex ARMS‐PCR method for the simultaneous genotyping of the circulating SARS‐CoV‐2 phylogenetic clades
Md. Tanvir Islam, A. S. M. Rubayet Ul Alam, Najmuj Sakib, Md. Shazid Hasan, Tanay Chakrovarty, Mohammad Tawyabur, Ovinu Kibria Islam, Hassan M. Al‐Emran, Mohammad Iqbal Kabir Jahid, M. Anwar Hossain
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.