Percorrer por autor "Sequeira, Z."
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- Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant virusesPublication . Nolasco, Gustavo; Sequeira, Z.; Soares, C.; Mansinho, A.; Bailey, A. M.; Niblett, C. l.PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan(TM), a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.
- Bacterial expressed coat protein: development of a single antiserum for routine detection of Citrus tristeza virusPublication . Sequeira, Z.; Nolasco, GustavoCitrus tristeza virus (CTV) causes one of the most important citrus diseases world-wide and has recently been detected in Portugal. Early diagnosis based on immunoenzymatic techniques requires significant amounts of reagents. This research describes the isolation of the coat protein gene of CTV, its expression in Escherichia coli as a fusion protein containing an N-terminal (His)6 region and its use to raise rabbit polyclonal antibodies. These antibodies could be used at dilutions higher than 1/80,000 at the detection stage in indirect ELISA tests and were also efficient for trapping the virus in ELISA and Immunocapture RT-PCR. These characteristics allowed the production of diagnostic kits based solely on this source of antibodies. A detection spectrum and sensitivity similar to that of a commercial polyclonal antibody kit was achieved.
- 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.