A visual, rapid, and sensitive detection platform for Vibrio parahaemolyticus based on RPA-CRISPR/Cas12a and an immunochromatographic test strip

Abstract

Vibrio parahaemolyticus is the primary species that causes vibriosis. In this study, a point-of-care detection method was developed for V. parahaemolyticus. The detection platform targeted the thermolabile haemolysin (tlh) gene of V. parahaemolyticus based on recombinant polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR/Cas) systems. The platform was combined with an immunochromatographic test strip (ICS) that enables low-cost, simple, and visual detection of V. parahaemolyticus. The detection limit was 2.5×102 fg/µL for plasmids and 1.4×102 CFU/mL for V. parahaemolyticus. In addition, V. parahaemolyticus in salmon sashimi could be detected at the concentration of 154 CFU/g without enrichment, and the entire detection time was ~30 min. After enrichment for 6 h, 2 CFU/g of V. parahaemolyticus could be detected. Consequently, the proposed RPA-CRISPR/Cas12a-ICS platform could detect V. parahaemolyticus in seafood intuitively, quickly, and sensitively, leading to a high practical application value.