Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees

A single-step multiplex RT-PCR was developed for the simultaneous and colou rimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf r oll virus, strawberry latent ringspot virus. Arabis mosaic virus, Olive lat ent-1 virus and Olive latent-2 virus) which infect olive trees. Six compati ble primer set for one-step RT-PCR amplification in a single closed-tube an d 3' digoxigenin labelled probes were designed in optimal, specific and con served regions. The method has been assessed with 195 Spanish field olive t rees, suggesting that similar to 1.5% of the tested material was infected b y Cucumber mosaic virus and 0.5'%, by Cherry leaf roll virus. This method s aves time and reagent costs compared with monospecific RT-PCR which needs s everal reactions for the same number of tests. Using colourimetric detectio n, 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. (C) 2001 Elsevier Science B.V. A ll rights reserved.