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.

自2012年以来，番茄叶子卷曲新德里病毒（ToLCNDV）是一种双歧杆菌病毒，影响地中海盆地的葫芦科和茄科作物。根据新的欧盟植物健康法规2016/2031，它已被归类为检疫性有害生物。为了获得可靠且特异性的检测病毒的诊断方法，设计了针对ToLCNDV基因组B成分BC1区的新引物和探针组，并根据报道的指南验证了实时扩增方案在EPPO标准PM 7/98（4）中。测试了该协议的分析灵敏度，分析特异性，选择性，可重复性和可再现性，从而为所有验证参数获得了一致的结果。该协议还适用于从其载体昆虫中检测ToLCNDV烟粉虱（Bemisia tabaci）（一种能够传播begomoviruses的粉虱），使用的是从单个有毒的成虫中提取的DNA。在昆虫载体中检测ToLCNDV可能是一种在大规模传播之前及早识别进口材料或新农业地区中病毒的有效方法。据我们所知，这是根据EPPO标准PM7 / 98（4）要求的验证参数对ToLCNDV进行验证的第一种方法。这种实时PCR可以成功地用于ToLCNDV的特异性检测，以满足植物健康新法规对植物检疫的要求。