Design and in silico validation of variant-specific
(3′-SNP) multiplex primers
A set of 15 primers (Table 1 ) was designed based on the ARMS
for differentiating six major clades of SARS-CoV-2: S, L, V, G, GH, and
GR. We designated here the L clade strains as the wild type and others
as mutants. For each clade apart from L, we selected a single
representative SNP variant, including 23403 A>G (p.D614G),
25563 G>T (p.Q57H), 28882 G>A (p.R203K), 26144
G>T (p.G251V) and 28144 T>C (p.L84S) from the
multiple co-evolving mutations of the clades (G, GH, GR, V and S,
respectively). For example, the S clade is deviated from the L clade by
two mutations: C8782T, T28144C. The ‘T’ or ‘C’ at 28144 positions was
rendered as wild (L clade) or mutant type variant (indicating S clade),
respectively. Details of other clade-specific mutations can be derived
from the GISAID site and this literature. As established for ARMS
technique, this specificity was directed towards the 3′-end of the
annealed primer-template (Fig. 1). The forward or reverse type-specific
primers were paired with counterpart reverse or forward primer. The
amplicons were simultaneously distinguished by their molecular weight
(bp) in multiplex PCR in different combinations. The positive
amplification of wild type targeting primers was determined as the L
type. The other types were determined based on the co-evolving mutation
at respective sites.
The primer sets were designed using Primer3Plus (Untergasser et al.,
2012) and Primer-BLAST (Ye et al., 2012) with the following stringent
parameters and standard PCR conditions: avoiding hypothetical primer
dimer (self or hetero) formation with less than -9 Kcal/mol, sized 18-22
nucleotide in length, Tm of (58-60)°C, (40-60)% GC content, G/C within
the last five bases, no repeat of four or more of any base, amplicon
size ranging from 200 to 600 bp, and avoiding hairpin loop structure.
The primer specificity against SARS-CoV-2 and other organisms was
checked by the Primer-BLAST. We performed the in silico PCR with
the primers in the UCSC genome browser (https://genome.ucsc.edu/).
Finally, the primer set was synthesized from the IDT company
(https://www.idtdna.com/).