Usefulness of an in vitro-transcribed RNA control for the detection and quantification of Yellow fever virus through real-time reverse transcription-polymerase chain reaction

[Display omitted] •A positive control plasmid DNA for molecular diagnostics of YFV was generated.•T7-driven transcription allowed the generation of a YFV positive control RNA.•The control RNA was successfully used for the detection and quantification of YFV through real-time RT-PCR.•The YFV construct is available for use with two recommended YFV detection assays. Unvaccinated individuals in endemic areas with proven enzootic transmission of Yellow fever virus are at risk of infection due to a dramatic shift in the epidemiology of the disease over recent years. For this reason, epidemiological surveillance and laboratory confirmation of cases have become mandatory. To develop and test a control RNA for YFV detection through real-time RT-PCR. A 437-bp insert containing the T7 promoter and the target sequences for two different in-house protocols was designed in the context of the pUC57 vector and obtained through gene synthesis. After T7-driven in vitro transcription, standard curves were developed for Log10 serial dilutions of the YFV control RNA with 8 replicates. A dynamic range of quantification of 10 orders of magnitude was observed with a limit of detection of 6.3 GCE/µL (95% CI, 2.6 to 139.4 GCE/µL). The plasmid construct is available for YFV molecular test validation on clinical, entomological, and epizootic samples.