Harmonization and validation of diagnostic protocols for detection of Plum pox virus.

Plum pox virus is one of the most detrimental pathogens of stone fruit trees, largely spread in many areas throughout 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 framework of the Project ARNADIA financed by the Italian Ministry of Agriculture, to set up a validated diagnostic protocol, officially approved and published at the national level. Different diagnostic methods, protocols and reagents were compared in five different laboratories, using the same target and non-target reference samples. ELISA, RT-PCR and real time RT-PCR protocols were selected and applied to determine their performance characteristics for validation under the standard ISO 17025. Sensitivity was determined performing experiments with seven serial dilutions of sample extracts for serological analysis and of total RNA extracts for molecular analysis. Specificity was determined assaying different infected samples, representative of the genomic and geographical variability of PPV, and non-target samples. Reproducibility was assessed through performance of the experiments by different laboratories. Repeatability will be established in the frame of a ring test among Italian phytosanitary laboratories. Results showed that the accuracy and sensitivity of ELISA and RT-PCR are comparable, whereas real time RT-PCR is recommended for testing symptomless samples.