Reversetranscription, real-time PCR assay for detection of Toscana virus

BackgroundThe arthropod-borne Toscana virus is a common cause of acute neurological infection in the Mediterranean basin. Recently, a new lineage, highly divergent from the Italian prototype, has been reported in Spain.ObjectiveWe describe a reversetranscription, real-time PCR assay for detection of both Toscana virus genotypes. The real-time PCR uses a TaqMan® probe and an internal control to identify false negative results.Study designA conserved region of the two known lineages of Toscana virus, located at the 3′ end of the small segment of their genomes, was chosen to design both the primers and the probe.ResultsThe sensitivity of the assay was 0.0158 TICD50 per reaction of Toscana virus, equivalent to seven copies of cDNA. No other phleboviruses or RNA viruses were amplified by this specific real-time PCR.ConclusionsThe assay seems to be sensitive, reliable and easy to be applied in the diagnosis of autochthonous and/or imported suspected cases of Toscana virus infection.