CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Abstract

<i>Vibrio parahaemolyticus</i> is one of the major pathogenic <i>Vibrio</i> species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE-RAA-CRISPR) for detecting <i>V. parahaemolyticus</i> in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for <i>V. parahaemolyticus</i>, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 10¹ CFU/mL in pure cultures and 7.3 × 10¹ CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE-RAA-CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.