Reverse transcription, real-time PCR assay for detection of Toscana virus.
BACKGROUND
The 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.


OBJECTIVE
We describe a reverse transcription, 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.


METHODS
A 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.


RESULTS
The sensitivity of the assay was 0.0158 TICD(50) 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.


CONCLUSIONS
The 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.
Pérez-Ruiz
Mercedes
M
Servicio de Microbiología, Hospital Universitario Virgen de las Nieves, Avda. Fuerzas Armadas, 2, 18014 Granada, Spain. mercedes.perez.ruiz.sspa@juntadeandalucia.es
Collao
Ximena
X
Navarro-Marí
José-María
JM
Tenorio
Antonio
A
D000818:Animals; D001483:Base Sequence; D002044:Bunyaviridae Infections / Q000175:diagnosis / Q000821:virology; D002555:Cerebrospinal Fluid / Q000821:virology; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D008587:Meningitis, Viral / Q000175:diagnosis / Q000821:virology; D008969:Molecular Sequence Data; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D029301:Sandfly fever Naples virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014709:Vero Cells
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral