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/).