Detection of grapevine fanleaf virus by dot-blot hybridization with biotin-labelled GFV-cDNA probe

The 5′and 3'-primers of 21 bp of GFV coat protein gene were synthesized according to the GFV-RNA sequence,The cDNA of GFV ccat protein gene was syntheeized with reverse transcriptase using3′-primer and GFV-RNA extracted from the Durified virus as template,Then the ds-cDNA was amplified by PCR technique employing GFV-RNA_2:ss-cDNA as templates and 5',3′-primers and ligated-with pBluesclpt ks.The ds-cDNA were transformed into Ecoli XLI-Blue cells,g the cDNA extracted from the recombinantclones of E. coli cells.The biotin-labelled ss,ds-probes have been used for the detection of purified GFV-RNA,GFV-infeted C,quivoa and 18 grapevine leaves by nucleic acid dot-blot hybridization,The minimal amount ofpurified GFV-RNA_2 which gave the visible signal was 1.5pg/test dot and the maximum dilution ofGFV-infected C,quinoa extract which gave visible signal was 1:40960;11 samples with typical symptoms were,petive and some of them could give the signal when they were diluted 1:400￣1:800.3samples out of 7 symptomeless leaves