Rapid Multiple Visual Detection of Alternaria alternata Causing Pear Black Spot via RPA-CRISPR/Cas12a

Alternaria alternata, the fungal pathogen primarily responsible for black spot disease in pears, is among the more destructive pathogens of the species (Pyrus spp.) worldwide as well as a major mycotoxin producer, posing significant threats to food safety and public health, particularly in under-resourced areas. Conventional detection methods, such as morphological characterization and PCR, are time consuming and lab dependent, limiting their application in on-site agricultural diagnostics. The present study aimed to develop a rapid, sensitive, and field-deployable RPA-CRISPR/Cas12a assay for the detection of A . alternata . The system was further optimized for point-of-care use by incorporating multiple accessible readouts, including fluorescence quantification, visual detection under UV/blue light, lateral flow strips, and poly(3-(3’-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) chromogenic reactions, making it highly suitable for those applications. The entire workflow, from simplified DNA extraction to visual interpretation of results, can be completed within 40 min. The optimized assay demonstrated high specificity for A . alternata and achieved a detection limit of 1 copy/μL per reaction, surpassing the sensitivity of conventional PCR/qPCR methods. Furthermore, field validation with 54 pear leaf and fruit samples showed complete concordance with PCR. This RPA-CRISPR/Cas12a platform combines specificity, high sensitivity, and flexible visual detection, eliminating the need for complex instrumentation and offering a field-deployable solution for the early, accurate diagnosis of pear black spot disease. • RPA-CRISPR/Cas12a enables rapid visual detection of Alternaria alternata in pear. • Multi-mode readout (fluorescence, UV/blue, LFS, PMNT) ensures clear interpretation. • Ultra-sensitive detection limit of 1 copy/μL, outperforming PCR/qPCR. • Field-deployable for on-site pear black spot diagnosis without sophisticated gear