Establishment of RPA-CRISPR/Cas12a-based visual detection of Vibrio chagasii

Vibrio chagasii is an emerging pathogen in crassostrea angulata aquaculture, causing significant economic losses and posing risks to food safety. Traditional detection methods, such as PCR, are time-consuming and rely on complex equipment, making them unsuitable for on-site rapid detection. Therefore, developing fast, sensitive, and simple detection methods is of great importance. To meet the need for rapid detection of V. chagasii, this study integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, aiming to establish real-time fluorescence detection method and test strip detection based on RPA-CRISPR/Cas12a. Specific fragments targeting the toxR gene of V. chagasii were designed, and RPA primers and crRNA sequences were optimized to ensure accurate gene amplification and identification. Upon recognition of the RPA-amplified target gene, the CRISPR/Cas12a system activated its trans-cleavage activity, cleaving biotin-modified single-stranded DNA fluorescent probes. Detection results were determined by the fluorescence brightness under blue light and the color development of the test strip. The experiment used multiple Vibrio species to verify the specificity of the detection methods and evaluated the sensitivity of the two detection methods through different concentrations of V. chagasii bacterial solution. The two methods were further compared with PCR using real samples. The established RPA-CRISPR/Cas12a-based fluorescence detection method and test strip detection demonstrated excellent specificity, accurately identifying V. chagasii, and the whole detection process could be completed within 1 h. Both methods achieved a detection limit of 100 CFU/mL, showing high consistency with PCR results (100% concordance). The RPA-CRISPR/Cas12a-based detection methods developed in this study are fast, sensitive, and easy to operate, providing a reliable technical solution for the on-site detection of V. chagasii. This study offers two novel, efficient, and convenient methods for the rapid detection of V. chagasii, contributing significantly to disease control in aquaculture and ensuring food safety.