RPA-mediated CRISPR/Cas12a-based assay for rapid and highly specific detection of soft rot-causing bacterial pathogen Dickeya fangzhongdai

Dickeya fangzhongdai is a virulent, cell wall-degrading soft rot pathogen within the Dickeya genus, causing economic losses to global agriculture. The rapid and accurate detection of this pathogen is crucial for effective management, highlighting the need for precise and rapid diagnostic methods. In this study, we developed a temperature-insensitive, rapid, and highly specific CRISPR/Cas12a-based assay to accurately detect D. fangzhongdai. The assay was designed using a unique region of the MFS transporter gene, from which 12 crRNA and RPA/PCR primers were developed. After screening all 12 crRNAs, crRNA-3 was selected due to its highest cleavage efficiency. The assay’s specificity was tested using an exclusivity panel consisting of all known Dickeya and Pectobacterium species, showing no cross-reactivity with any strain except D. fangzhongdai. It can detect genomic DNA as low as 10 pg. This assay can detect the target region in under 10 minutes (5 min for RPA, 5 min for CRISPR/Cas12a cleavage and detection). While the assay was optimized to work at 37°C, it also showed robust performance at room temperature and even in hand palm, demonstrating its temperature insensitivity and potential for field applications. Results also indicated that crRNA does not bind to the target if there is a difference of more than one nucleotide, thereby preventing cleavage and detection. This characteristic makes the CRISPR/Cas12a-based assay highly specific and robust. This assay has potential applications in routine diagnostics, biosecurity, microbial forensics, and disease epidemiology.