Figure 2. Validation of new real-time RT-PCRs for differentiation between vaccine and wild-type YFV. A) Effects of target competition on YFV real-time RT-PCRs. Mean cycle threshold (Ct) values are plotted against IVT concentrations. Triplicates were tested for each datum point. B) Validation of the assays with clinical matrices. Spiked viruses were vaccine strain 17D and the American genotype 2 wild-type strain BOL88/1999. RNA purification was performed using the MagNA Pure 96 Viral NA Small Volume Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. C) Clinical validation. Clinical specimens (serum, liver, whole blood, and plasma) from 11 YFV-infected patients were tested. RNA was extracted using the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher, São Paulo, Brazil) and serial dilutions of the RNA were tested using the new assays and a YFV reference assay (12). Viral loads were determined for clinical specimens using a commercially available quantitative real-time RT-PCR (Bio Gene Research Yellow Fever PCR kit; Bioclin, Minas Gerais, Brazil), following the manufacturer´s instructions. Standard curves and sample copies per millileter were calculated using an in-house IVT standard. IVT, in vitro transcript; RT-PCR, reverse transcription PCR; YFV, yellow fever virus.