Detection of GFLV in Xiphinema index with DNA

Poster sessions
108 Detection of GFLV in Xiphinema index with DNA-based fluorescent probes
Mariella F INETTI -S IALER 1 and Aurelio C IANCIO2,∗
1 Dipartimento
di Protezione delle Piante, Università degli Studi, Bari, Italy
per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Via Amendola 165/A, 70126 Bari, Italy
∗ [email protected]
2 Istituto
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 RNA2 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.