Rapid and Sensitive Detection of Apple stem pitting virus in Apple Trees Through RNA Amplification and Probing with Fluorescent Molecular Beacons

ABSTRACT Currently, detection of Apple stem pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in ‘Golden Delicious’ and ‘Gravenstein’ apple trees was compared thr...