*25 µL real-time RT-PCR reactions were performed using the Superscript III one-step RT-PCR system with Platinum Taq polymerase (Thermo Fisher Scientific, Darmstadt, Germany). Target genomic domain positions according to GenBank reference genome NC_002031. Reactions were set up with 5 μL of RNA; 12.5 μL of 2× reaction buffer; 0.4 μL of a 50 mM magnesium sulfate solution (Superscript III one-step RT–PCR system with Platinum Taq polymerase kit, Thermo Fisher Scientific); 1 μg of nonacetylated bovine serum albumin; and 1 µL enzyme. Single-target assay reactions contained 400 nM forward primer, 600 nM reverse primer, and 280 nM of each probe. Dual-target assay reactions contained 400 nM of each primer and 220 nM of each probe. RT-PCR, reverse transcription PCR.
†Probes are labeled with either fluorescein amidite (FAM) or Yakima Yellow (YAK) at the 5′-end and a Black Hole Quencher (TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) at the 3′-end. Primer concentrations were optimized using the YFV vaccine strain IVT and the wild-type YFV IVT. Amplification involved 50°C for 15 min, followed by 95°C for 3 min and 45 cycles of 95°C for 15 s and 58°C for 30 s with fluorescence read at the 58° annealing/extension step on a LightCycler 480 thermocycler (Roche, Basel, Switzerland).
‡H = A/C/T, M = A/C, R = A/G, W = A/T, Y = C/T.