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Updated: Jan 14, 2026
Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes
Published on: July 27, 2018
An optimized DNA extraction protocol for reliable PCR-based detection and characterization of grapevine flavescence
Marco Carli1,2, Athos Pedrelli3,4, Alessandra Panattoni1
1Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, Pisa, 56124, Italy.
Background:
Flavescence dorée (FD) is one of the most damaging grapevine diseases in Europe, caused by the quarantine-listed Grapevine flavescence dorée phytoplasma (FDp). Given the absence of resistant cultivars and curative treatments, effective disease control relies on early and accurate FDp detection. PCR-based diagnostics are the gold standard, but their accuracy depends on DNA extraction quality. Grapevine tissues contain PCR inhibitors like polysaccharides and polyphenols, complicating DNA isolation. While CTAB methods yield high-quality DNA, they are time-consuming, and commercial kits provide purer but often lower DNA yields at high costs. A rapid and optimized DNA extraction method for FDp detection is urgently needed.
Results:
We developed the "HotShot Vitis" (HSV) method, a modified HotSHOT protocol optimized for grapevine tissues. HSV was benchmarked against the CTAB method and a commercial silica membrane kit. Although HSV showed limitations in DNA quantification due to buffer composition, it efficiently extracted DNA suitable for amplifying the grapevine trnL-F gene and detecting FDp by two qPCR assays. DNA extracted by HSV also supported molecular typing and sequencing of FDp 16 S rRNA and map genes, performing comparably to CTAB and the commercial kit. Importantly, HSV reduced the extraction time to about 30 min, significantly faster than the CTAB (2 h) and kit (40 min) methods.
Conclusions:
HSV is a fast, reliable, and chemically low-risk DNA extraction method for FDp detection and characterization in grapevine. Its efficiency and simplicity make HSV ideal for large-scale diagnostics and early disease management.
