Quality assessment and validation of digital PCR (dPCR) for grapevine virus diagnosis

Grapevine is a highly economically important crop in Mexico. However, it can be affected by several pathogens, including viruses that can cause significant crop losses. It is important to identify early the infected plants to manage the disease correctly and prevent economic losses. Traditional detection methods have drawbacks, such as limited sensibility and accuracy. Digital PCR (dPCR) is an innovative method that claims to be more sensitive and reproducible than the routine method for virus identification: quantitative PCR (qPCR). This study assesses reverse transcription dPCR (RT-dPCR) as a method for the detection and quantification of RNA grapevine viruses focusing on grapevine virus A (GVA), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), and grapevine Pinot gris virus (GPGV). This assessment was performed using positive controls and comparing the limit of detection (LoD) results of RT-dPCR against the results obtained by RT-qPCR. ANOVA results showed that the PCR technique (RT-dPCR or RT-qPCR) and the virus (GVA, GLFV, GPGV, and GLRaV-3) were statistically significant in the results of the comparison of LoD. Furthermore, the replicates were non-significant according to ANOVA, showing high repeatability in both RT-qPCR and RT-dPCR. Tukey test demonstrated that RT-dPCR is significantly more sensitive than RT-qPCR, with a statistically reliable difference of 95% trust, especially in low-viral-load viruses such GPGV, which detection showed also to be statistically different than the other viruses. Additionally, a field study was performed to identify the presence or absence of each virus in 45 grapevine samples evaluated with RT-qPCR and RT-dPCR. Several false negative results were generated by RT-qPCR, which only reported positive results to 62.5% of the GVA infected samples, 85.7% of the samples contaminated with GLRaV-3, 38.9% of GPGV positive samples and for GFLV only 12.5% of the infected samples were identified. These results confirm the effectiveness of RT-dPCR as a sensitive method for RNA virus detection in grapevine, enabling early diagnosis and optimal management of viral infections in grapevine crops.

https://orcid.org/0000-0002-4736-8019