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

Grapevine black-foot disease is a destructive trunk disease with a complex pathogen composition that often involves mixed and latent infections, making timely field diagnosis challenging. To improve rapid field detection, we developed a rapid, sensitive, and low instrument-dependent nucleic acid assay. The assay integrates recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a for the detection of Ilyonectria and Dactylonectria, two genera associated with grapevine black-foot disease. Conserved regions of the histone H3 and β-tubulin genes were selected for the design of specific RPA primers and corresponding CRISPR RNAs (crRNAs) for Ilyonectria and Dactylonectria, respectively. A workflow integrating RPA, Cas12a-mediated recognition, and lateral flow assay (LFA)-based visualization was established. The reaction conditions were optimized to enhance amplification efficiency and Cas12a recognition stability. Specificity was evaluated using DNA from target and non-target fungi, and sensitivity was determined using serially diluted templates. Under optimized conditions, the assay detected Ilyonectria DNA at concentrations as low as 3.6 ng/μL within 1 h at 39 °C. For Dactylonectria, the detection limit reached 80 fg/μL within 50 min at 41 °C. No cross-reactivity was observed. The LFA strips exhibited positive and negative bands within minutes, enabling rapid visual interpretation. This RPA-CRISPR/Cas12a-LFA system provides a rapid, visually interpretable approach for detecting selected grapevine black-foot disease-associated species in China. The workflow reduces the requirement for specialized thermocycling and fluorescence detection equipment during amplification and readout, following DNA extraction.