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 4.5×105 CFU/mL. The detection sensitivity of real-time fluorescent PMA-LAMP were 4.5×100 CFU/mL and PMA-qPCR were 4.5×101 CFU/mL. In addition, the correlation coefficients (R2) 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.