Development and application of a sensitive droplet digital PCR (ddPCR) for the detection of infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis, caused by the infectious spleen and kidney necrosis virus (ISKNV), has damaged the economy of the world fish industry. Droplet digital PCR (ddPCR) is a novel, sensitive, accurate and absolute quantitation method that does not require a standard curve. In this study, we established a sensitive ddPCR method to rapidly detect and quantify ISKNV DNA. The established method is highly specific to ISKNV and does not cross-react with other iridoviruses. The detection limit of the ddPCR was found to be 1.5 copies/mu L, which is much lower than the 34 copies/mu L determined for TaqMan real-time PCR (qPCR). This indicated that the sensitivity of the ddPCR assay was 20-fold higher than the sensitivity of the qPCR assay. We explored the feasibility of ddPCR to detect ISKNV from 23 fry samples (mandarin fish, Siniperca chuatsi) and compared the data with qPCR, in terms of sensitivity and accuracy. The detection results for fry samples showed that the positive detection rate of ddPCR (65.22%) was higher than that of qPCR (30.43%). In conclusion, the ddPCR method shows superiority for detection in samples with low ISKNV viral loads, which will facilitate the surveillance of sources and transmission routes of ISKNV.