Overcoming sensitivity limitations: RT-dPCR for accurate grapevine virus diagnosis

Grapevine, an economically important crop in Mexico, is vulnerable to viral pathogens that can induce yield reductions. Timely detection of infected plants is paramount for effective disease management and mitigating economic losses. Traditional diagnostic methods, such as quantitative PCR (qPCR), often suffer from limitations in sensitivity and accuracy. This study evaluates the efficacy of reverse transcription digital PCR (RT-dPCR) for the detection and quantification of four key RNA grapevine viruses: grapevine virus A (GVA), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), and grapevine Pinot gris virus (GPGV). By comparing the limit of detection (LoD) between RT-dPCR and RT-qPCR using positive controls, we demonstrated through ANOVA that both the PCR technique and the specific virus significantly influenced LoD values. Notably, replicates exhibited high repeatability across both methods. Tukey's post-hoc analysis confirmed that RT-dPCR exhibited significantly higher sensitivity than RT-qPCR, particularly for low-titer viruses like GPGV. Further, a field study of 45 grapevine samples revealed a high incidence of false-negative results with RT-qPCR. Specifically, RT-qPCR detected only 62.5% of GVA, 85.7% of GLRaV-3, 38.9% of GPGV, and 12.5% of GFLV infected samples. These findings underscore the superior sensitivity of RT-dPCR for RNA virus detection in grapevine, providing a powerful tool for early diagnosis and optimized disease management.