title: Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees.
abstract: A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.
authors.affiliation: Departamento de Protección Vegetal y Biotecnología, Laboratorio de Virología e Inmunología, Instituto Valenciano de Investigaciones Agrarias, Apartado oficial, 46113 Moncada Valencia, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Bertolini
authors.forename: A
authors.initials: A
authors.lastname: Olmos
authors.forename: M C
authors.initials: MC
authors.lastname: Martínez
authors.forename: M T
authors.initials: MT
authors.lastname: Gorris
authors.forename: M
authors.initials: M
authors.lastname: Cambra
mesh_terms: D003124:Colorimetry / Q000379:methods; D017931:DNA Primers; D019684:Magnoliopsida / Q000821:virology*; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014197:Trees / Q000821:virology*
chemical_list: D017931:DNA Primers