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Vibrio parahaemolyticus  is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of  V. parahaemolyticus  based on  tlh ,  tdh ,  ureR , and  orf8  in food samples using single intact cells. The targets  tlh  and  ureR  were labeled with 6-Carboxyfluorescein (FAM), and the targets  tdh  and  orf8  were labeled with 5’-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (2 4 ) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of “rain” and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of  V. parahaemolyticus  including pandemic group strains and could be applied in the differentiation of  V. parahaemolyticus  in a wide variety of samples.

frontiers in microbiology

A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Based on Single Intact Cells