Application of a lyophilized CRISPR/Cas12a and RPA assay for rapid detection of Actinobacillus pleuropneumoniae

• Combines RPA and CRISPR/Cas12a, significantly enhancing detection sensitivity and specificity. • Introduces lyophilization, improving stability and shelf-life, and eliminating cold chain dependence. • Enables straightforward, naked-eye visualization of results, simplifying rapid identification. Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ), a major pathogen in swine, causes severe respiratory illnesses, resulting in substantial economic losses for the global pork industry. Traditional detection methods are labor-intensive and time-consuming, highlighting the need for rapid, sensitive, and point-of-care diagnostic approaches. This study presents the development of an optimized CRISPR/Cas12a system integrated with a recombinase polymerase amplification (RPA) method targeting the apxIVA gene of A. pleuropneumoniae . The RPA-CRISPR/Cas12a assay demonstrated high specificity across various bacterial genomic DNA samples, with a detection limit as low as 3.0 copies/µL, validated under both excitation light and non-excitation light conditions. To address the challenge of reagent stability and eliminate cold chain dependency, we successfully stabilized assay reagents through lyophilization, incorporating saccharide-based protective agents. The optimized lyophilization parameters ensured the long-term stability and efficacy of the lyophilized reagents, offering a significant improvement over liquid storage. This method offers a new, efficient, and convenient method for rapid diagnosis of swine diseases.