Detection and absolute quantitation of Broad bean wilt virus 1 (BBWV-1) and BBWV-2 by real time RT-PCR

Broad bean wilt virus 1 (BBWV-1) and BBWV-2 are the two most significant viruses in the genus Fabavirus, causing damage to many economically important agricultural crops worldwide. A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using two TaqMan®MGB probes was developed for sensitive and specific detection and quantitation of BBWV-1 and BBWV-2. Primers and probes were designed from conserved sequence stretches to detect all isolates of each virus. Standard curves using RNA transcripts identical to both TaqMan®MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (103–1010 RNA molecules). RT-qPCR was assayed with genetically divergent BBWV-1 and BBWV-2 isolates from different plant hosts and countries, and was used to evaluate the temporal accumulation of BBWV-1 RNA in two plant hosts.

► Detection and quantitation of a wide spectrum of Broad bean wilt virus (BBWV-1) or BBWV-2 isolates. ► Design of Taqman®MGB probes for specific detection and quantitation of BBWV-1 or BBWV-2. ► Very sensitive detection (100 viral RNA molecules per 1 ng of total RNA). ► Analysis of temporal accumulation of BBWV-1 in Vicia faba and Chenopodium quinoa plants.