Abstract
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.
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