Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated...

Area of Science:

• Plant Pathology
• Molecular Virology
• Agricultural Diagnostics

Background:

• Grapevine leafroll disease (GLD) significantly impacts viticulture, particularly in South Africa.
• Grapevine leafroll-associated virus type 3 (GLRaV-3) is a primary causal agent and is widespread in South African vineyards.
• Current detection methods like ELISA are time-consuming and less sensitive for field applications.

Purpose of the Study:

• To develop a rapid, sensitive, and field-deployable diagnostic assay for GLRaV-3.
• To provide an alternative to existing methods for efficient grapevine disease management.

Main Methods:

• Development of a single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay.
• Incorporation of a simple RNA extraction protocol and hydroxy napthol blue as a visual indicator.
• Validation through direct comparison with ELISA and nested PCR methods.

Main Results:

• The RT-LAMP assay successfully detected GLRaV-3 under isothermal conditions (60 °C) within 2 hours.
• Positive results were indicated by a clear color change (violet to sky blue).
• The assay demonstrated sensitivity comparable to nested PCR and outperformed ELISA in speed and simplicity, with the ability to detect one positive sample in a pool of 50.

Conclusions:

• The developed RT-LAMP assay offers a rapid, sensitive, and user-friendly method for GLRaV-3 detection.
• This assay presents a viable alternative to ELISA for field-based diagnostics, aiding in effective GLD management.
• The method's efficiency and pooling capability can streamline vineyard disease surveillance.