Vibrio parahemolyticus and Salmonella spp.are the notorious pathogens in seafood,which poses a great potential threat to public health.In this work,a real-time multiplex PCR combined with propidium monoazide (PMA) was developed for simultaneous quantification of viable V.parahemolyticus and Salmonella spp.in raw shrimp.In the series of adjustments and optimizations,this novel method was proved as a powerful tool which could detect as low as 102 CFU/g of both strains in raw shrimp without from 106 CFU/g dead cells.Furthermore,this optimized assay has been successfully employed to develop the inactivation model of V.parahemolyticus and Salmonella spp.by ultra-high pressure (HHP) and acidic electrolyzed water (AEW) treatment.In conclusion,the novel multiplex PMA-qPCR not only could be a rapid and effective diagnostic tool for the monitoring of viable V.parahemolyticus and Salmonella spp.in seafood,but also a reliable tool for construction of microbial inactivation model.