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

• Published in: Journal of virological methods Vol. 96; no. 1; pp. 33 - 41
• Main Authors: Bertolini, Edson, Olmos, Antonio, Martı́nez, M.Carmen, Gorris, Marı́a Teresa, Cambra, Mariano
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.07.2001; Amsterdam Elsevier; New York, NY
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Colorimetry - methods; Cucumber mosaic virus; Detection; DNA Primers; Fundamental and applied biological sciences. Psychology; Hybridisation; Magnoliopsida - virology; Microbiology; Multiplex RT-PCR; Olea europaea; Olive latent virus 1; Plant Diseases - virology; Plant Viruses - genetics; Plant Viruses - isolation & purification; Primer design; Reverse Transcriptase Polymerase Chain Reaction; RNA Viruses - genetics; RNA Viruses - isolation & purification; Sensitivity and Specificity; Techniques used in virology; Trees - virology; Virology; Multiplex RT-PCR; Olea europaea; Primer design; Detection; Hybridisation; Olive latent virus 1; Cherry; RNA; Rosaceae; Comoviridae; Cherry leaf roll virus; Dicotyledones; Cucumber mosaic virus; Angiospermae; Bromoviridae; Tree; Necrovirus; Nepovirus; Oleaceae; Fragaria; Tombusviridae; Cucurbitaceae; Cucumis sativus; Oleavirus; Strawberry latent ringspot virus; Virus; Polymerase chain reaction; Latent; Prunus avium; Cucumovirus; Spermatophyta; Arabis mosaic virus; Olive latent virus 2
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(01)00313-5

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.