Development and comparative analysis of RT-PCR and Real-Time (TaqMan®) PCR techniques for the detection of olive viruses
Résumé
Real-time- and Multiplex-TaqMan RT-PCR assays were developed to detect single and multiple infections of five olive viruses, i.e. Arabis mosaic virus, Cherry leafroll virus, Strawberry latent ring spot virus, Olive latent ring spot virus and Olive leaf yellowing-associated virus. The newly designed primers and probes were constructed upon viral nucleotide sequences alignments of different isolates from Genbank, taken into consideration all degeneracies encountered at probes and primers sites. After validating the new techniques on 20 different isolates, both assays showed to be highly efficient and more sensitive than conventional RT-PCR (from 103 to 106 times, according to the virus). The quantitative real-time RT-PCR assay developed in this study to quantify RNA-targets of the five viruses in infected crude sap material was able to detect 0.25 fg up to 75pg viral RNA.
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In Multiplex-Real Time PCR all the fiveviruses in study were efficiently detected simultaneously. These two diagnostic techniques can be useful to control and limit olive virus spread and for supporting quarantine and certification programs.