A Rapid Recombinase Polymerase Amplification-CRISPR/Cas12a Assay for Detecting Grapevine Black-Foot Pathogens

Area of Science:

• Plant Pathology
• Molecular Biology
• Biotechnology

Background:

• Grapevine black-foot disease poses a significant threat to vineyards.
• Complex and latent infections make early field diagnosis difficult.
• Existing diagnostic methods can be time-consuming and require specialized equipment.

Purpose of the Study:

• To develop a rapid, sensitive, and field-deployable nucleic acid assay for grapevine black-foot disease pathogens.
• To detect key genera, Ilyonectria and Dactylonectria, associated with the disease.
• To simplify the diagnostic workflow, reducing reliance on complex instrumentation.

Main Methods:

• Developed a nucleic acid assay integrating recombinase polymerase amplification (RPA) and CRISPR-Cas12a technology.
• Designed specific RPA primers and CRISPR RNAs targeting conserved histone H3 and β-tubulin genes.
• Integrated RPA with Cas12a-mediated recognition and lateral flow assay (LFA) for visual readout.

Main Results:

• The assay detected Ilyonectria DNA at concentrations as low as 3.6 ng/μL within 1 hour at 39 °C.
• The detection limit for Dactylonectria reached 80 fg/μL within 50 minutes at 41 °C.
• Lateral flow assay strips provided clear positive/negative results within minutes, demonstrating high specificity.

Conclusions:

• The developed RPA-CRISPR/Cas12a-LFA system enables rapid, visual, and sensitive detection of grapevine black-foot disease pathogens.
• This assay significantly reduces the need for specialized laboratory equipment, facilitating field application.
• The system offers a promising tool for timely diagnosis and management of grapevine trunk diseases.