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

Abstract

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 (R) 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 (R) MGB probes enabled absolute quantitation, with a wide dynamic range and high sensitivity (10(3)-10(10) 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. (C) 2011 Elsevier B.V. All rights reserved.