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.