Development and Validation of a Degenerate Primer and LNA Probe Set for Rapid, Reliable, and Sensitive Detection of grapevine rupestris stem pitting-associated virus (Foveavirus rupestris; GRSPaV) in Certification Programs

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a significant pathogen in certification programs, causing stem pitting in the trunks of grafted vines. Nevertheless, it remains latent in ungrafted varieties and rootstocks. Given its status as a certification agent, rapid, reliable, and sensitive detection of GRSPaV is of great importance. However, the identification of genetically distinct variants of GRSPaV has made reliable diagnosis using the current primer pair employed in certification analyses challenging, leading to false-negative results. In this study, degenerate primers and an LNA probe set were designed using 25 genomic sequences from five different GRSPaV strain groups to achieve rapid, reliable, and sensitive detection. Optimization studies were conducted using the Roche LightCycler® Nano Real-time PCR system. Real-time RT-PCR results carried out via 41 grapevine varieties and 81 rootstock samples demonstrated that the newly designed degenerate primer pair and LNA probe set yielded positive results in 37 out of 122 vine samples, whereas the existing primer pair detected only 5 positive samples. The findings indicate that the designed degenerate primer and LNA probe set can be successfully utilized for the rapid, reliable, and sensitive detection of GRSPaV in certification analyses. Given the high genetic variability of viruses like GRSPaV, robust detection requires continuously updated primers, rigorous strain monitoring, and skilled diagnosticians since a universally perfect, future-proof primer remains an unattainable ideal.