A New HiPlex Amplicon Sequencing Approach for the Detection of Grapevine Leafroll-Associated Virus 3 and Grapevine Red Blotch Virus in Grapevines

Grapevine leafroll-associated virus 3 (GLRaV3) and grapevine red blotch virus (GRBV) are two major pathogens associated with significant economic losses, requiring reliable and sensitive diagnostic tools for rapid identification. Current RT-qPCR and qPCR assays are widely used in certification programs; however, the risk of false-negative results due to the genetic diversity of plant viruses is always a concern. Here, we present a new targeted HiPlex assay designed for the detection of GLRaV3 and GRBV. A custom primer panel containing 98 oligonucleotides targeting GLRaV3 and 14 targeting GRBV was evaluated across 45 different grapevine accessions. HiPlex diagnostic performance metrics were evaluated up to the 1:1000 dilution level. The HiPlex assay detected both viruses in 30 ng of total nucleic acids (TNA) and at 1:10 and 1:100 dilutions, exhibiting a strong inverse correlation between read counts and Ct values. Analysis of reads from qPCR-negative samples allowed the determination of a positivity cutoff of 5000 reads, which provided high specificity while maintaining sensitivity. The results presented here support the application of HiPlex amplicon sequencing for the detection of GLRaV3 and GRBV and provides a foundation for future expansion of the primer panel to additional grapevine viruses. To our knowledge, this is the first study evaluating multiplex PCR-based amplicon sequencing specifically for grapevine virus diagnostics.