Grapevine Fleck Virus: Molecular Detection and Investigation of Strain Variability
Specific PCR primers were designed to amplify the coat protein gene of GFKV to use in sequence comparisons of different isolates. Amplification by regular PCR was attempted two times and failed. Finally we designed another pair of primers, internal to the first set and used in nested RT-PCR and successfully amplified the majority of the genome (592 nt out of 672 nt for the full length of the coat protein gene). This nested RT-PCR was tried on 23 different GFKV isolates and it was successful on 17 of them. The sequence comparison among these 17 isolates revealed that they were quite homogeneous and had similarities between 95% to 99.5%. We have found that nested RT-PCR is more sensitive than regular PCR. Although it requires an additional step to complete, but in our preliminary experiments it was shown to be tens of times more sensitive than regular RT-PCR. Based on its sensitivity and reproducibility, the nested RT-PCR will have a great potential for use in testing grape materials for virus detection.