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 ∼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.