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 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.
Bertolini
E
E
Departamento de Protección Vegetal y Biotecnología, Laboratorio de Virología e Inmunología, Instituto Valenciano de Investigaciones Agrarias, Apartado oficial, 46113 Moncada Valencia, Spain.
Olmos
A
A
Martínez
M C
MC
Gorris
M T
MT
Cambra
M
M
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*
D017931:DNA Primers