Abstract 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/μL, which is much lower than the 34 copies/μL 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.

中文翻译：

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用于检测传染性脾肾坏死病毒的灵敏液滴数字PCR（ddPCR）的开发与应用

摘要 传染性脾肾坏死病毒（ISKNV）引起的传染性脾肾坏死已经破坏了世界渔业经济。液滴数字 PCR (ddPCR) 是一种新颖、灵敏、准确和绝对的定量方法，不需要标准曲线。在这项研究中，我们建立了一种灵敏的 ddPCR 方法来快速检测和量化 ISKNV DNA。已建立的方法对 ISKNV 具有高度特异性，并且不会与其他虹彩病毒发生交叉反应。发现 ddPCR 的检测限为 1.5 拷贝/μL，远低于 TaqMan 实时 PCR (qPCR) 确定的 34 拷贝/μL。这表明 ddPCR 检测的灵敏度比 qPCR 检测的灵敏度高 20 倍。我们探讨了 ddPCR 从 23 个鱼苗样本（鳜鱼、Siniperca chuatsi) 并在灵敏度和准确性方面将数据与 qPCR 进行了比较。对鱼苗样品的检测结果表明，ddPCR的阳性检出率（65.22%）高于qPCR（30.43%）。综上所述，ddPCR 方法在检测 ISKNV 病毒载量较低的样本方面表现出优越性，这将有助于 ISKNV 来源和传播途径的监测。