One Real-Time Fluorescent Loop-Mediated Isothermal Amplification Combined with Propidium Monoazide for Detection of Viable Vibrio Parahaemolyticus in Seafood

Abstract

Vibrio parahaemolyticus is one of the crucial foodborne pathogens in the world. At present, the rapid detection methods for V. parahaemolyticus cannot distinguish between dead and viable cells and false positive results may occur. In this study, one rapid and accurate method that combines Propidium Monoazide (PMA) with real-time fluorescent loop-mediated isothermal amplification (LAMP) using SYTO-16 dye was developed to detect viable cells of V. parahaemolyticus. LAMP amplification was performed on specific primers designed for toxR gene of V. parahaemolyticus and the concentration of PMA and the maximum concentration of dead cells were optimized. The results showed that the concentration of PMA was 10 µM and PMA could efficiently treat dead cells up to 4.5×10 5 CFU/mL. The detection sensitivity of real-time fluorescent PMA-LAMP were 4.5×10 0 CFU/mL and PMA-qPCR were 4.5×10 1 CFU/mL. In addition, the correlation coefficients (R 2) is 0.9992, indicating that SYTO-16 dyed real-time fluorescent PMA-LAMP assay can be used for quantification with high sensitivity. This method exhibits high specificity and sensitivity, can be used as an effective tool for rapid detection of V. parahaemolyticus and a scientific basis to follow the effect of the pathogen infection on growth of cultured seafood.

Quantitative Detection of Viable but Nonculturable Vibrio parahaemolyticus in Frozen Bivalve Molluscs

“…Only clam samples were positive for VBNC V. parahaemolyticus . While there are numerous studies on the use of PMA-qPCR on artificially contaminated seafood samples, including shrimp, crab, fish, and shellfish [ 23 , 62 , 64 , 66 , 96 ], few of them have addressed the efficacy of these methods for detecting V. parahaemolyticus in marketed seafood. Yu et al [ 96 ], in a comparative study using real-time fluorescent PMA-LAMP and PMA-qPCR on 139 fishery product samples, including cod, grilled croaker, dried squid, and shrimp, reported a positivity of 2.16% for both methods.…”