Harmonization and validation of diagnostic protocols for the detection of Plum pox virus
Pasquini, G.; Bianco, P.A.; Boscia, D.; Casati, P.; Digiaro, M.; Faggioli, F.; Palmisano, F.; Poggi-Pollini, C.; Rubies, C.; Barba, M.
Journal of Plant Pathology
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Plum pox virus is one of the most detrimental pathogens of stone fruit trees, largely spread in many areas all over the world. It is included in the EPPO A2 Quarantine pathogens list and must be submitted to the international and national quarantine requirements. The high importance of the harmonization of the diagnostic procedures, the contribution to greater transparency during the diagnosis of regulated pests and the need of the resolution of disputes among trading partners suggested, in the frame of the Italian Project ARNADIA financed by the Ministry of Agriculture, to set up a validated diagnostic protocol, officially approved and published at national level. Different diagnostic methods, protocols and reagents have been compared in five different laboratories, using the same target and non-target reference samples. Protocols of ELISA, RT-PCR and real time RT-PCR have been selected and developed to determine the performance characteristics for the validation under the standard ISO 17025. The sensitivity has been determined performing experiments with seven serial dilutions of sample extracts in serological analysis and of total RNA extracted in molecular analysis. The specificity has been determined assaying different infected samples, representing the genomic and geographical variability of PPV, and non-target samples. The reproducibility has been obtained performing the experiments with different operators. The repeatability will be established in the frame of a ring test among Italian phytosanitary laboratories. The results show that the accuracy and sensitivity of ELISA and RT-PCR are comparable, whereas real time RT-PCR is recommendable with asymptomatic samples.