The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR.
Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Beet necrotic yellow vein virus (BNYVV). The two assays designed were a broad-spectrum one that detected RNA 2 from all types and a second designed to detect types containing RNA 5. The assays were validated against a range of different isolates from Europe and the Far East. These real-time assays were compared to a conventional RT-PCR assay for the detection of RNA 5. Sensitivity comparisons showed that for the detection of RNA 5, TaqMan was 10,000 times more sensitive than the conventional RT-PCR assay. Further improvements were made to the test procedure by using post-ELISA virus release (VR), as an alternative to RNA extraction. This significantly increased the speed of processing samples and reduced the staff input required, allowing the TaqMan assay to be used routinely as part of an annual survey of UK field samples.
Harju
V A
VA
Central Science Laboratory (CSL), Sand Hutton, York YO41 1LZ, UK.
Skelton
A
A
Clover
G R G
GR
Ratti
C
C
Boonham
N
N
Henry
C M
CM
Mumford
R A
RA
D027461:Beta vulgaris / Q000821:virology*; D010935:Plant Diseases / Q000821:virology; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D012367:RNA, Viral; D019914:Taq Polymerase