Establishment and Application of RPA-CRISPR/Cas12a-based Visual Detection of Botrytis cinerea

Botrytis cinerea, the causal agent of gray mold in facility vegetables, is characterized by a wide host range and rapid post-infection spread, often leading to severe economic losses. This paper aims to establish an early and precise detection method for gray mold before its visible symptoms appear, and to provide technical support for the early monitoring and targeted control of gray mold in facility vegetable cultivation.

A conserved gene sequence (BC1G_05726) from B. cinerea strain B05.10 was used as the target. Specific recombinase polymerase isothermal amplification (RPA) primers and CRISPR/Cas12a crRNAs were designed and screened. Key parameters including reaction time and the concentration ratio of Cas12a to crRNA were systematically optimized to establish a rapid visual detection method combining RPA and CRISPR/Cas12a. Fluorescence signals were visualized using a portable blue light transilluminator. The specificity, sensitivity, and applicability of the developed method were evaluated using complex environmental samples (infested soil, seeds, symptomatic plants, and air samples).

The optimal crRNA (crRNA-1) and primer pair (RPA-bc-F/R) were selected. The optimized detection conditions were as follows: RPA reaction at 37 ℃ for 20 min, followed by CRISPR/Cas12a reaction with final concentrations of 200 nmol·L^-1 for Cas12a and 50 nmol·L^-1 for crRNA for another 20 min. The entire detection process was completed within 40 min. The sensitivity assay revealed a detection limit of 10^-4 ng·μL^-1 of genomic DNA of B. cinerea, which was 100-fold more sensitive than conventional PCR. Specificity testing showed that only B. cinerea produced positive fluorescence signals, while none of the other 14 tested fungal and bacterial species or the negative control generated any signal, indicating excellent specificity of the assay. In application tests, the method successfully detected B. cinerea strains from different hosts. After artificial inoculation on cucumber leaves, the pathogen was stably detected as early as 1 d post-inoculation, preceding symptom appearance. Dynamic monitoring of air samples in the facility environment demonstrated that B. cinerea could be detected in the air at 60 h post-inoculation, at which time typical disease lesions had not yet developed on the plants, validating the early warning capability of this method.

The RPA-CRISPR/Cas12a-based visual detection method established in this study enables rapid, sensitive, and specific detection of B. cinerea under isothermal conditions without requiring complex instrumentation, and the results can be read by the naked eye. This method is suitable for on-site detection of the pathogen in soil, seed, plant, and air samples.