Harmonization and validation of diagnostic protocols for the detection of grapevine viruses covered by phytosanitary rules

The Italian Ministry of Agriculture funded the Finalized Project "ARNADIA", aimed at producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest and, among them, grapevine viruses. In this framework, the "Working group ARNADIA - grapevine viruses (WG)", composed of 8 Universities and Research Bodies, 3 accredited Private Laboratories, one Plant Health Service and one Association of Grapevine Nurseries was established. Moreover, 5 additional Italian Plant Protection Services took part in an inter-laboratory ring test.The aim of the WG was to produce reference and validated serological and molecular protocols allowing for the harmonization of the diagnosis of 8 grapevine viruses, namely, Grapevine leafroll-associated virus-1,-2,-3, (GLRaV 1, 2, 3) Grapevine virus A (GVA), Grapevine virus B (GVB), Arabis mosaic virus (ArMV), Grapevine fanleaf virus(GFLV) and Grapevine fleck virus (GFkV). Accordingly, the validation of the protocol consists in the evaluation of the processes aimed at determining their fitness for the particular use, and the validation of the assay yields test results that identify the presence of a specific target. The parameters that influence the capability of the test result to accurately predict the sample's infection status are: diagnostic sensitivity (ability of the used method to detect the presence of the pathogen in the samples truely infected by the pathogen in question - true positive) and diagnostic specificity (ability of the used method NOT to detect the presence of the pathogen in samples not infected by the pathogen in question - true negative). Other parameters that must be considered and which determine the efficiency of a protocol are: the analytical sensitivity (the smallest amount of infectious entities that can be identified by the diagnostic method), repeatability or accordance (degree of conformity of the results obtained in replications of the process, made at short time intervals, using the same reference sample and in the same working conditions i.e. equipment, operator, laboratory) and reproducibility or concordance (degree of conformity of the results obtained using the same method with the same reference samples in different laboratories). We reported the parameters obtained in the validation of a serological (ELISA) and molecular (Multiplex RT-PCR) protocols for the diagnosis of eight grapevine viruses.