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.