Percorrer por autor "Teixeira Santos, Margarida"
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- Genetic variants of Grapevine leafroll-associated virus 2 infecting Portuguese grapevine cultivarsPublication . Fonseca, Filomena; Esteves, Filipa; Teixeira Santos, Margarida; Brazão, João; Eiras-Dias, José EduardoGenetic variability of 19 isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) from Portuguese grapevine cultivars was characterized by sequencing the entire capsid protein (CP) gene of the virus. Global phylogenetic analysis of the CP gene, which included nucleotide sequences obtained in this study and complete homologous sequences from GenBank, showed segregation of GLRaV-2 variants from Portuguese isolates into three major phylogroups (PN, 93/955 and H4). The novelty of these phylogenetic results is the evidence of well-supported subdivision within H4 as well as within PN, with subgroup PN3 composed exclusively of variants from a Portuguese isolate. These findings and the genetic analysis of global phylogroups indicate demographic expansion, mainly within PN and 93/955. Because the existence of a mixture of variants from different phylogroups was detected in some of the isolates, a typification assay based on reverse transcription reaction followed by polymerase chain reaction and restriction fragment length polymorphism analysis, was developed to complement molecular detection assay of the virus. This protocol discriminates variants from the phylogroups identified in this study, and is appropriate for routine testing for GLRaV-2.
- Molecular data mining to improve antibody-based detection of grapevine leafroll-associated virus 1 (GLRaV-1).Publication . Esteves, Filipa; Teixeira Santos, Margarida; Eiras-Dias, José Eduardo; Fonseca, FilomenaTesting for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.