Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water.

Pseudomonas fluorescens is the main spoilage bacterium in milk, and its proliferation is one of the factors leading to the deterioration of the quality of raw milk. In this study, a rapid detection system for P. fluorescens was developed based on recombinase-aided amplification combined with a test strip (RAA-TS), which contained a double test line (DTL) targeting the virulence gene aprX of P. fluorescens and the housekeeping gene gyrB of Pseudomonas. Visual observation could detect gyrB (50 CFU/mL) and aprX (250 CFU/mL) within 90 min, including sample pretreatment and RAA reaction and detection steps. No cross-reactions were observed with Pseudomonas or other bacteria (n = 19). The quantitative detection limits (LOD) of gyrB and aprX for P. fluorescens in milk were 37 CFU/mL and 233 CFU/mL, respectively. Compared with polymerase chain reaction-agarose gel electrophoresis (PCR-AGE), the sensitivity of the developed RAA-TS-DTL system was increased by approximately four times. Furthermore, it could detect live P. fluorescens in milk when combined with optimized sample pretreatment by propidium monoazide (PMAxx). Its consistency with the traditional culture method in the detection of P. fluorescens spiked in milk samples (n = 25) was 100%. The developed RAA-TS-DTL had the advantages of high accuracy and short time consumption. Thus, it provides a new way or tool for the rapid screening or detection of P. fluorescens in milk.