title: Generic RT-PCR tests for detection and identification of tospoviruses.
abstract: A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.
authors.affiliation: National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O. Box 9102, 6700HC, Wageningen, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Hassani-Mehraban
authors.affiliation: National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O. Box 9102, 6700HC, Wageningen, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Westenberg
authors.affiliation: National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O. Box 9102, 6700HC, Wageningen, The Netherlands.
authors.forename: J T J
authors.initials: JT
authors.lastname: Verhoeven
authors.affiliation: National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O. Box 9102, 6700HC, Wageningen, The Netherlands.
authors.forename: B T L H
authors.initials: BT
authors.lastname: van de Vossenberg
authors.affiliation: Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Kormelink
authors.affiliation: National Plant Protection Organization, National Reference Centre, Geertjesweg 15, P.O. Box 9102, 6700HC, Wageningen, The Netherlands. Electronic address: j.w.roenhorst@nvwa.nl.
authors.forename: J W
authors.initials: JW
authors.lastname: Roenhorst
mesh_terms: D019251:Nucleocapsid / Q000235:genetics; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D010944:Plants / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017865:Tospovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
keywords: Bunyaviridae; Clade-specific primers; Diagnostics; Nucleocapsid gene; Plant virus