A multi-channel CRISPR-based method for rapid, sensitive detection of four diseases of <i>Brassica rapa</i> in the field

Pathogens significantly restrict the production of <i>Brassica rapa</i> (<i>B. rapa</i> L. ssp. Pekinensis), with climate change and evolving planting patterns exacerbating disease prevalence. Multichannel rapid diagnostic methods in the field can facilitate the early detection and control of diseases in <i>B. rapa</i>. Here, we established a multichannel lateral flow biosensor (LFB) combined with a CRISPR/Cas12a cleavage assay for the simultaneous detection of four <i>B. rapa</i> diseases. Key innovations of this study include: (1) High specificity and sensitivity, down to pathogen concentrations of 1.5 pg/μl-due to the optimization of crRNA secondary structure: the more stable the crRNA, the higher its detection sensitivity. (2) Optimized visual detection parameters. We identified ideal concentration ratios for the visual fluorescence detection system: 50 nM Cas12a, 50 nM crRNA, and 500 nM ssDNA fluorescent probe. Furthermore, the optimal concentrations of components on the LFB detection system were 3 μl SA-GNPs, 500 nM ssDNA test strip probe, 0.5 mg/ml biotin-BSA as the test line, and 1 mg/ml anti-FITC as the control line. (3) Field-Ready Cas-AIRPA Platform. We developed the on-site Cas-AIRPA platform for the simultaneous detection of <i>B. rapa</i> pathogens by combining rapid nucleic acid extraction and a four-channel lateral flow biosensor (4-LFB), which quickly provides disease-related information through a specific 2D barcode. Analysis of <i>B. rapa</i> samples in the field confirmed the suitability of the Cas-AIRPA platform for rapid (~25 min) and simultaneous on-site detection of four diseases of <i>B. rapa</i>. This platform can also be adapted to detect other plant diseases in the field.