An integrated INAA-CRISPR/Cas-LFIA approach for the detection of plant pathogens: A review

Plant pathogens and pests cause substantial economic losses in agriculture worldwide. To reduce crop damage, researchers have increasingly focused on developing compact, efficient detection systems. The integration of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease technologies has significantly advanced plant pathogen diagnostics. Recent progress has led to the development of rapid, on-site disease detection platforms. In particular, combining isothermal nucleic acid amplification (INAA) techniques with CRISPR/Cas systems offers high sensitivity and specificity, with results visually interpreted using lateral flow immunoassay (LFIA) strips. Among INAA methods, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) show great promise for field-based applications. CRISPR/Cas proteins bind specifically to amplified products from LAMP or RPA and cleave labeled reporter molecules through their collateral (trans-cleavage) activity. These cleaved reporters are detected by LFIA strips, producing a visible red signal. This review summarizes recent advancements in integrating INAA, CRISPR/Cas, and LFIA technologies for rapid and reliable detection of plant pathogens.