title: Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan(®)MGB probes.
abstract: A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.
authors.affiliation: Instituto Valenciano de Investigaciones Agrarias, 46113 Moncada, Valencia, Spain.
authors.forename: D E
authors.initials: DE
authors.lastname: Debreczeni
authors.forename: S
authors.initials: S
authors.lastname: Ruiz-Ruiz
authors.forename: J
authors.initials: J
authors.lastname: Aramburu
authors.forename: C
authors.initials: C
authors.lastname: López
authors.forename: B
authors.initials: B
authors.lastname: Belliure
authors.forename: L
authors.initials: L
authors.lastname: Galipienso
authors.forename: S
authors.initials: S
authors.lastname: Soler
authors.forename: L
authors.initials: L
authors.lastname: Rubio
mesh_terms: D000818:Animals; D002212:Capsicum / Q000821:virology; D015342:DNA Probes; D032302:Datura / Q000821:virology; D005838:Genotype; D007303:Insect Vectors / Q000821:virology*; D007313:Insecta / Q000821:virology*; D018551:Solanum lycopersicum / Q000821:virology; D010935:Plant Diseases / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D017865:Tospovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D015342:DNA Probes; D012367:RNA, Viral; D019914:Taq Polymerase