Figure 1. Schematic workflow of ARMS-based multiplex PCR assays
for the identification of SARS-CoV-2 clades. The upper portion of the
figure showed the concept of clade as described in the GISAID with a
comprehensive genomic visualization. The lower segment is dedicated to
the overall workflow and the primer design.
Figure 2. Strategy and validation of ARMS-based multiplex PCR
assays . (I) Single-variant specific PCRs with the respective
amplicons in bp at the bottom of each gel image, showing the variants.(II) Multiplex PCR assays, containing PCR products for
different clade combinations: duplex for 26144 G>T
(p.G251V) and, 28144 T>C (p.L84S) variants at an annealing
temperature of 60°C; triplex for the variants 23403 A>G
(p.D614G), 25563 G>T (p.Q57H) and, 28882 G>A
(p.R203K) at 57°C; quadruplex for the variants 23403 A>G
(p.D614G), 25563 G>T (p.Q57H), 28882 G>A
(p.R203K) and, 28144C T>C (p.L84S) at 56°C. SARS-CoV-2
positive sample, GC 46.003 was used as a representative to perform the
PCRs for (I) and (II). (III) Validation of the multiplex PCR
assays, containing the identical settings for duplex, triplex and
quadruplex PCRs with GC 44.201 (denoted as SR-1), GC 88.025 (denoted
SR-2), GC 90.175 (denoted SR-3) and GC 92.172 (denoted as SR-4) as
representatives to display the reproducibility of the assays. SARS-CoV-2
negative sample, GC 116.09 was used as (-ve) control for the comparison.
‘GC’ indicates the Genome Center identification number generated at the
Genome Center of Jashore University of Science and Technology for
COVID-19 suspected patients. WT, wild type band; M, Mutant band.
100-1000bp marker was used in the first lane of the 1% agarose gels.
The primers are listed in Table 1.