Development and validation of a specific real-time PCR protocol for the detection of tomato leaf curl New Delhi virus

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting cucurbitaceous and solanaceous crops in the Mediterranean basin since the 2012. It has been categorized as quarantine pest according to the new EU Plant Health Regulation 2016/2031. In order to obtain a reliable and specific diagnostic method to detect the virus, a new primers and probe set, targeting the BC1 region of the component B of the ToLCNDV genome, was designed and the real-time amplification protocol validated according to the guidelines reported in the EPPO standard PM 7/98 (4). The protocol was tested for analytical sensitivity, analytical specificity, selectivity, repeatability and reproducibility obtaining consistent results for all the validation parameters. The protocol is also suitable for detecting ToLCNDV from its vector, the insect Bemisia tabaci (a whitefly able to transmit begomoviruses) using DNA extracted from a single viruliferous adult. Detection of ToLCNDV in insect vector could be an efficient way to early identify the virus in imported material or in new agricultural areas, before its massively spreading. To our knowledge, this is the first validated detection method for ToLCNDV according to the validation parameters required by the EPPO standard PM7/98 (4). This real-time PCR can be successfully applied to the specific detection of ToLCNDV satisfying the phytosanitary requirements of the new legislation on plant health.