Duplex droplet digital PCR method for the detection of Enterocytozoon hepatopenaei and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease

Enterocytozoon hepatopenaei (EHP) and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND) are two of the diseases that have frequently infected farmed shrimp in recent years, causing great economic losses to the shrimp industry worldwide. In this study, we established a sensitive and accurate duplex droplet digital PCR (ddPCR) method that can simultaneously detect and quantify the two pathogens simultaneously. The results showed that the ddPCR methods could detect EHP and VPAHPND specifically. The sensitivity levels of ddPCR for EHP and VPAHPND were 2.3 copies/mu l and 4.6 copies/mu l, respectively, which were 10-fold higher than the sensitivity of the qPCR assay and showed good reproducibility. Twenty-six suspected diseased shrimp samples were used for practical determination. For EHP, the detection rates of ddPCR and qPCR were 53.84% and 42.31%, respectively; for VPAHPND, the detection rates of ddPCR and qPCR were both 23.08%. The results indicated that the ddPCR method shows superiority for detection in samples with low viral loads, which will facilitate monitoring of the source and transmission of EHP and VPAHPND and will help control shrimp epidemic disease.