Tracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders viral genotyping in low-income countries. Here, we propose a rapid, simple, and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. 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 in the primer concentration (0.2-0.6 µM) and annealing temperature (56-60°C) of PCR using a 3-5 ng/µl cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentrations for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is a sample, cost-effective, and convenient that can successfully discriminate against the circulating phylogenetic clades of SARS-CoV-2.

The SARS-CoV-2 strain of the coronavirus is responsible for the current COVID-19 pandemic, with an ongoing toll of over 5 million infections and 333 thousand deaths worldwide within the first 5 months. Insight into the phylodynamics and mutation variants of this strain is vital to understanding the nature of its spread in different climate conditions. The incidence rate of COVID-19 is increasing at an alarming pace within subtropical Southeast Asian nations with high temperatures and humidity. To understand this spread, we analyzed 60 genome sequences of SARS-CoV-2 available in GISAID platform from 6 Southeast Asian countries. Multiple sequence alignments and maximum likelihood phylogenetic analyses were performed to analyze and characterize the non-synonymous mutant variants circulating in this region. Global mutation distribution analysis showed that the majority of the mutations found in this region are also prevalent in Europe and North America, and the concurrent presence of these mutations at a high frequency in Australia and Saudi Arabia indicate possible transmission routes. Unique spike protein and non-structural protein mutations were observed circulating within a localized area. We divided the circulating viral strains into 4 major groups and 2 sub-groups on the basis of the most frequent non-synonymous mutations. Strains with a unique set of 4 co-evolving mutations were found to be circulating at a high frequency within India, specifically, group 2 strains characterized by two co-evolving NS mutants which alter in RdRp (P323L) and spike protein (D614G) common in Europe and North America. These European and North American variants (Nextstrain clade A2) have rapidly emerged as dominant strains within Southeast Asia, increasing from a 0% presence in January to an 85% presence by May 2020. These variants may have an evolutionary advantage over their ancestral types and could present the largest threat to Southeast Asia for the coming winter.