Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
Department of Fish and Wildlife Sciences and the Aquaculture Research Institute, University of Idaho, Moscow, ID 83843-1136, USA.
J Virol Methods. 2018 Mar;253:38-42. doi: 10.1016/j.jviromet.2017.12.008. Epub 2017 Dec 26.
Infectious spleen and kidney necrosis virus (ISKNV) has been recognized as the causative agent of the most serious disease in cultured mandarin fish, Siniperca chuatsi, in China. Disease outbreaks have resulted in substantial losses to the aquaculture industry. Currently, reliable laboratory detection and identification methods are available for this virus. However, rapid detection methods applicable for on-site diagnosis of this infectious agent are unavailable. To address this need, a nearly instrument-free, cost-effective and simple detection method was developed and optimized and incorporates cross priming amplification coupled with vertical flow visualization for rapid identification of ISKNV (ISKNV-CPA-VF). Results show that cross circulation amplification targeting the conserved region of the major capsid protein (MCP) regiment of the ISKNV genome had a sensitivity 10 times greater than traditional PCR at 64 °C within 60 min. The optimized concentration of dNTPs and the concentration for Mg were 1.0 mmol/L and 10 mmol/L, respectively. No cross-reactions with other viruses or bacteria were observed. When combined with the nucleic acid strip detection technology, visual detection of ISKNV amplified products was realized within 3-5 min following amplification. The simplicity and nearly instrument-free method for this ISKNV-CPA-VF assay shows great potential for on-site diagnostics of ISKNV infection in Siniperca chuatsi.
传染性脾肾坏死病毒（ISKNV）已被认为是中国养殖鳜鱼最严重疾病的病原体。疾病爆发导致水产养殖业遭受重大损失。目前，已经有可靠的实验室检测和鉴定方法可用于检测这种病毒。然而，目前还没有适用于现场诊断这种传染性病原体的快速检测方法。为了满足这一需求，开发并优化了一种几乎无需仪器、具有成本效益且简单的检测方法，该方法结合了交联引物扩增和垂直流可视化，用于快速鉴定 ISKNV（ISKNV-CPA-VF）。结果表明，针对 ISKNV 基因组主要衣壳蛋白（MCP）regiment 保守区域的交联循环扩增在 64°C 下 60 分钟内的灵敏度比传统 PCR 高 10 倍。最佳的 dNTP 浓度和 Mg 浓度分别为 1.0mmol/L 和 10mmol/L。未观察到与其他病毒或细菌的交叉反应。与核酸条带检测技术相结合，在扩增后 3-5 分钟内即可实现对 ISKNV 扩增产物的可视化检测。ISKNV-CPA-VF 检测方法的简单性和几乎无需仪器的特点使其具有在现场诊断鳜鱼 ISKNV 感染的巨大潜力。
