A highly versatile real-time quantitative RT-PCR method and sampling strategies for the accurate detection of citrus yellow vein clearing virus

Citrus yellow vein clearing virus (CYVCV) is the causal agent of an emerging disease representing a potentially high-impact threat for citrus production. Despite remaining outside Europe for decades, CYVCV has now expanded towards two important European citrus producers, Italy and, more recently, Spain. The presence of this virus in the EPPO region represents a current threat with unpredictable and potentially devastating consequences for European citriculture. Therefore, urgent protective measures need to be taken to prevent CYVCV spread and minimize its impact. Diagnostics is a key measure in the management of viral diseases, highlighting the need for harmonized methods suitable for reliable routine detection of the currently known CYVCV diversity. In this study, an inclusive, efficient and highly sensitive real-time RT-qPCR for the detection of CYVCV in plant material and transmission vectors has been developed and validated according to EPPO standards. Moreover, the validated method has been successfully adapted to both PCR digital platforms, that allow high-sensitive absolute quantitative detection, essential in the diagnostics at low viral concentrations; and PCR portable tools, that can be applied in a real diagnostic context for on-site detection. This versatility combines standard validated performance, absolute sensitive quantitation and real on-site detection. The study has also addressed sampling strategies to support reliable molecular diagnostic performance. Our results represent an improvement in the detection of CYVCV to be applied in epidemiological studies and different real diagnostic contexts for the containment of this important citrus pathogen.