A droplet digital PCR method(ddPCR)for sensitive detection and quantification of Carassius auratus herpesvirus(CaHV)

Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.