Novel Single Primer Isothermal Amplification Method for the Visual Detection of Vibrio parahaemolyticus

Abstract

As a typical halophilic bacterium, Vibrio parahaemolyticus (V. parahaemolyticus) is regarded as one of the marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. Herein, a novel single primer isothermal amplification (SPIA) technique which was modified with SYBR Green II was successfully developed for the visual detection of V. parahaemolyticus. Interestingly, the visual superiority of SPIA can be achieved by fluorescence or precipitate to analyze the target pathogens in the seafood samples. The experimental results showed that all of V. parahaemolyticus strains were positive by using the novel SPIA assay, whereas the non-V. parahaemolyticus counterparts were negative. Furthermore, the limit of detection (LOD) of the SPIA assay was 5.7 CFU/mL in pure culture and 42 CFU/g in raw oysters by the visual fluorescence, respectively. Compared with the standard method, the developed SPIA approach achieved the satisfactory results with the characteristics of 100% sensitivity, 98.21% specificity, and 99.17% accuracy in the detection of 241 seafood samples. The developed SPIA method has been demonstrated as a rapid, specific, sensitive, and visual tool for the detection of V. parahaemolyticus, which could be adopted for routine surveillance of food-borne pathogens in the seafood samples.