This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats: Yersinia pestis, Bacillus anthracis, Brucella spp., Burkholderia pseudomallei, and Francisella tularensis. parahaemolyticus in a wide variety of samples.īiothreat agents pose a huge threat to human and public health, necessitating the development of rapid and highly sensitive detection approaches. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. 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. 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. For better results, the sample hot lysis time and the cycle number were optimized. Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (24) clusters based on fluorescence amplitude were obtained. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5’-Hexachlorofluorescein (HEX). parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide.
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