Abstract: To meet the demand for rapid detection of Vibrio parahaemolyticus in aquatic products, a rapid one-tube detection method based on recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/CRISPR-associated protein 12a (Cas12a) was established and optimized. This method utilized RPA primers and CRISPR RNA (crRNA) specifically designed for the ToxR gene of V. parahaemolyticus. The specificity and sensitivity of this method were analyzed, and it was used to test artificially contaminated samples. Results indicated that under optimized conditions (final crRNA concentration 100 nmol/L, Cas12a:crRNA concentration ratio 0.5:1, and reporter probe:Cas12a concentration ratio 2.2:1), the detection process could be completed within 30 min at 37 ℃. The method demonstrated excellent specificity with no cross-reactivity to common pathogens. It also exhibited high sensitivity, with a detection limit of 102 CFU/mL for pure cultures of V. parahaemolyticus and 1.5 CFU/mL for artificially contaminated samples. In summary, the one-tube RPA-CRISPR/Cas12a assay offered the advantages of high sensitivity, strong specificity, and operational simplicity, providing a reliable technical approach for high-throughput rapid detection of V. parahaemolyticus in aquatic products.

Key words: recombinase polymerase amplification; CRISPR/Cas12a; one-tube detection; Vibrio parahaemolyticus; aquatic product safety