Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD)

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla(CARB-17) gene is an intrinsic beta-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla(CARB-17) gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non -V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg2+, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63oC for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non -V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 x 10(-4) ng/mu l, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

Automated summary

Vibrio parahaemolyticus is a significant foodborne pathogen causing gastroenteritis in humans, with the bla(CARB-17) gene being a novel species-specific genetic marker. The LAMP-LFD assay developed in this study showed high specificity for detecting V. parahaemolyticus, with successful detection of the pathogen in contaminated and natural food samples. This method displayed a sensitivity 100-fold higher than conventional PCR, making it a suitable diagnostic tool for detecting V. parahaemolyticus in aquatic foods.