Multiplex PCR assays for simultaneous identification of the variants
Four sets (duplex, triplex, quadruplex and pentaplex) of multiple variant-specific reactions were arranged for simultaneous detection of a clade. A duplex PCR was performed by using a mix of 26144 G>T(p.G251V) and 28144 T>C (p.L84S) variant specific primer pairs viz. NS3_26144_F-NS3_26144_wR (wild type)/NS3_26144_F-NS3_26144_mR (mutant) and NS8_28144_F-NS8_28144_wR (wild type)/ NS8_28144_F-NS8_28144_mR (mutant), respectively while mixing for wild types and the mutants in separate PCR tubes. A triplex PCR assay was performed by using a blend of primer pairs viz. S_23403_wF-S_23403_R (wild type primers)/ S_23403_mF- S_23403_R (mutant primers), NS3_25563_w1F-NS3_25563_1R (wild type primers)/ NS3_25563_m1F-NS3_25563_1R (mutant), and N_28882_F-N_28882_wR (wild type)/ N_28882_F-N_28882_mR specific to 23403 A>G (p.D614G), 25563 G>T (p.Q57H) and 28882 G>A (p.R203K) SNP variants respectively. Quadruplex and pentaplex PCR assays were further performed in similar manner. The pentaplex consisted the mix for all five SNP variants, On the other hand, the quadruplex contained the variants of triplex plus either 26144 G>T (p.G251V) or 28144 T>C (p.L84S), thus making two different subsets. Among the 24 samples, one was used as a representative of all sets of multiplexes and reproducibility (described in more detail below) was checked over the rest of the samples.