Detection of GFLV in Xiphinema index with DNA-based fluorescent probes

A PCR based method was applied to the detection of grapevine fanleaf virus (GFLV). The virus was amplified from specimens of its vector Xiphinema index, collected in a grapevine orchard at Palagiano, Italy. The detection was carried out with a real-time fluorescent RT-PCR assay. A set of primers was designed for the GFLV RNA-2 amplification. A 2500 bp fragment was amplified by RT-PCR, from X. index adults and juveniles, cloned and sequenced. We compared the obtained GFLV sequences with those available in GenBank and two regions were selected for virus detection and/or strain identification. A fluorescent Scorpion probe was designed to amplify a 100 bp fragment within the conserved region of the cp gene. A 21 bp conserved motif at position 2855 was used as probe target. An additional region with strain-specific nucleotide variations at position 2502 was used to discriminate between the Palagiano and other GFLV strains. Specific oligos, constructed on the basis of GenBank sequences AF304015 and X16907, were used as controls. Furthermore, a set of three strain-specific molecular beacons was designed on this region and used with the amplified DNA or oligos. The successful detection of the probe targets was shown by fluorescent signals emitted during amplification or under UV excitation. The Scorpion probe proved to be useful in virus detection. Due to their single-base mismatch sensitivity, the molecular beacons appeared suitable for specific strain recognition. The potential of these technologies in the study of transmission and vector epidemiology are briefly discussed.