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