Digital Polymerase Chain Reaction as a Sensitive Diagnostic Tool: Detection and Quantification of Grapevine Fleck Virus in Grapevine

Grapevine fleck virus (GFkV) is a graft-transmissible pathogen that threatens grapevine production in rootstocks and hybrid cultivars. Its early and accurate detection is essential for certifying virus-free planting materials. This study developed and validated a reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR) assay for the specific and absolute quantification of GFkV in grapevine tissues. The assay demonstrated high specificity, showing no cross-reactivity with other major grapevine viruses. Meanwhile, RT-ddPCR exhibited approximately 10-fold higher sensitivity than RT-quantitative PCR (RT-qPCR), with a detection limit of 0.73 copies/μl. Both RT-ddPCR and RT-qPCR exhibited high linearity (R2=0.9997 and 0.9983, respectively). In field validation, GFkV was detected in 84.8% (56/66) of the samples via RT-ddPCR, whereas RT-qPCR detected the virus in 53.0% (35/66) of the samples. These results indicate that RT-ddPCR is a highly sensitive and reliable method for GFkV detection in grapevines.