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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.
Plant Methods
|October 17, 2025
Summary
A new "HotShot Vitis" DNA extraction method rapidly detects Flavescence dorée phytoplasma in grapevines. This fast and reliable technique aids in early disease management for this damaging grapevine disease.
Area of Science:
- Plant Pathology
- Molecular Biology
- Agricultural Science
Background:
- Flavescence dorée (FD) is a destructive grapevine disease caused by Grapevine flavescence dorée phytoplasma (FDp).
- Effective FDp control hinges on early detection due to lack of resistant cultivars and treatments.
- Existing DNA extraction methods (CTAB, commercial kits) have drawbacks like time consumption, high cost, or lower DNA yield, necessitating a better approach.
Purpose of the Study:
- To develop and evaluate a rapid, optimized DNA extraction method for Flavescence dorée phytoplasma detection in grapevine.
- To benchmark the new method against established CTAB and commercial kit protocols.
Main Methods:
- Development of the "HotShot Vitis" (HSV) method, a modification of the HotSHOT protocol for grapevine tissues.
- Comparative analysis of HSV against CTAB and a commercial silica membrane kit for DNA extraction efficiency and downstream application suitability.
Main Results:
- The HSV method significantly reduced DNA extraction time to approximately 30 minutes, compared to 2 hours for CTAB and 40 minutes for the commercial kit.
- DNA extracted using HSV was suitable for amplifying grapevine genes and detecting FDp via qPCR assays.
- HSV facilitated molecular typing and sequencing of FDp genes, performing comparably to conventional methods.
Conclusions:
- "HotShot Vitis" (HSV) offers a fast, reliable, and chemically low-risk DNA extraction solution for FDp detection and characterization in grapevines.
- The method's efficiency and simplicity are advantageous for large-scale diagnostics and prompt disease management strategies.
