Large Scale Evaluation of Primers for Diagnosis of Rupestris Stem Pitting Associated Virus-1 |
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Authors: | G Nolasco A Mansinho M Teixeira Santos C Soares Z Sequeira C Sequeira PK Correia OA Sequeira |
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Institution: | (1) Universidade do Algarve, UCTA, Campus de Gambelas, 8000 Faro, Portugal;(2) Universidade do Algarve, UCTA, Campus de Gambelas, 8000 Faro, Portugal;(3) Estação Agronómica Nacional, Quinta do Marques, 2780 Oeiras, Portugal |
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Abstract: | 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. |
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Keywords: | PCR rupestris stem pitting sensitivity negative predictive value double-stranded RNA |
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