Extended Data Fig. 1: A Single Nucleotide Polymorphism (SNP) assay using minor groove binder (MGB)-based TaqMan probes for detecting wild-type (A388) or mutant (V388) genes in various conditions.
A SNP assay was developed for detecting wild-type (A388) or mutant (V388) HA genes in various conditions utilizing a set of Minor Groove Binder (MGB)-based TaqMan probes: VIC-labeled probe detecting the wild-type (A388) and a FAM-labeled probe detecting the mutant (V388). The SNP assay was validated using a mixed viral genome from the 2009 H1N1pdm wild-type (A388) and the mutant (V388) viruses. Viral RNA was mixed in varying ratios from 10:0 (0% mutant virus) to 0:10 (100% mutant virus). For the validation of two-step SNP assay for analyzing nasal wash samples from the human challenge study, mixed viral RNA was diluted to represent varying viral loads of (a) 105.5 TCID50/ml, (b) 104.0 TCID50/ml, and (c) 103.0 TCID50/ml. Prepared viral RNA was analyzed by the two-step SNP assay (see Methods). Blue and red bars indicate the ∆Rn value of wild-type and mutant virus, respectively. Graphs show mean ∆Rn value and standard deviation from 4 independent experiments (a-c, n = 4). For the validation of one-step SNP assay used to analyze selection dynamics in vitro, mixed viral RNA was diluted to represent varying viral loads of (d) 107.0 TCID50/ml, (e) 105.5 TCID50/ml, and (f) 104.0 TCID50/ml. Prepared viral RNA was analyzed by the one-step SNP assay (see Methods). Blue bars indicate the threshold cycle (Ct) values representing the amount of wild-type (A388) virus. Red bars represent the amount of mutant (V388) virus. Graphs show mean Ct value and standard deviation from 3 independent experiments (d-f, n = 3). Results show that the SNP assay reliably detects minor population, either wild-type or mutant, existing as low as 10% across the various viral loads.
