Nested RT-PCR for detection of sandfly fever virus, serotype Toscana, in clinical specimens, with confirmation by nucleotide sequence analysis.
A single tube, reverse transcriptase/polymerase chain reaction (RT-PCR) was developed and evaluated for detecting a 400-bp product of the small RNA of sandfly fever virus, serotype Toscana (TOS). For more sensitive detection of genomic TOS RNA, a nested PCR amplifying a 243-bp cDNA within the RT-PCR product was established. Nucleotide sequence analysis of first- and second-round PCR products using the dideoxy cycle sequencing technique confirmed a previously published sequence of the TOS reference strain (ISS. Phl.3). By nested PCR, genomic TOS RNA was amplified from two consecutive sera taken 3 and 7 weeks after the onset of illness in one patient, and from CSF of a second patient obtained at the onset of meningitis. Authenticity of amplified PCR products was confirmed by nucleotide sequence analysis, revealing a sequence identical to the TOS reference strain. RT-PCR and nested PCR are useful for laboratory diagnosis and studies of the molecular epidemiology of TOS infection.
Schwarz
T F
TF
Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University, Munich, Germany.
Jäger
G
G
Gilch
S
S
Nitschko
H
H
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D006801:Humans; D008587:Meningitis, Viral / Q000097:blood / Q000821:virology*; D008969:Molecular Sequence Data; D010217:Phlebotomus Fever / Q000097:blood / Q000821:virology*; D016856:Phlebovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D012703:Serotyping; D014709:Vero Cells
D012367:RNA, Viral