Browsing by Author "Sequeira, O. A."
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- Large scale evaluation of primers for diagnosis of rupestris stem pitting associated virus-1Publication . Nolasco, Gustavo; Mansinho, A.; Santos, M. T.; Soares, C.; Sequeira, Z.; Sequeira, C.; Correia, P. K.; Sequeira, O. A.The unavailability of adequate immunological reagents has prevented the use of ELISA for the diagnosis of rupestris stem pitting disorder of grapevines. In this work, the performance of five primer pairs for broad-scale detection of rupestris stem pitting associated virus-1 by RT-PCR using ds-RNA templates was compared and contrasted with biological indexing. The virus was widespread among the budwood of 35 Portuguese grapevine varieties assayed, with a prevalence of 85%. The biological assay proved to be unreliable as an index of infection due to the high number of false negatives. Five sets of primers were assayed and compared by means of their relative sensitivity and negative predictive value. The primer pair specific for the coat protein gene was excluded because of the difficulty in identifying the specific amplified product. From the other four primer pairs, those specific for the helicase domain of the putative polymerase gene had the highest sensitivity and negative predictive value. However, a high confidence in the assay, as desirable for a certification scheme, could not be obtained by the sole use of this primer pair. An additional pair should be used in a separate or in a multiplex RT-PCR reaction.
- Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat proteinPublication . Nolasco, Gustavo; Santos, C.; Santos, M. T.; Cortez, I.; Fonseca, Filomena; Petrovic, N.; Boben, J.; Pereira, A. M. N.; Sequeira, O. A.Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.