Mu Quanling, Ding Cunbao, Xie Ying, Zhen Xi, Zhang Jiaming, Yu Yakun
College of Life Sciences, North China University of Technology, Caofeidian District, Tangshan, Hebei 063210, China.
College of Life Sciences, North China University of Technology, Caofeidian District, Tangshan, Hebei 063210, China.
J Virol Methods. 2025 Jul;336:115156. doi: 10.1016/j.jviromet.2025.115156. Epub 2025 Apr 12.
This study focuses on three viruses affecting farmed shrimp, including White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), and Taura Syndrome Virus (TSV). Specific primers and probes were designed by their respective conserved gene fragments to establish a triple-RPA-LFS detection method that simultaneously detects WSSV, IHHNV, and TSV. Seven pathogens and healthy shrimp tissues were collected to conduct specificity tests. This method can specifically amplify the gene fragments of WSSV, IHHNV, and TSV, while no fragments were amplified from the muscle tissues of healthy shrimp or other pathogens, indicating strong specificity. The reaction system was optimized, and specificity and sensitivity were validated. Sensitivity tests were conducted using a continuous dilution plasmid method, determining that the detection sensitivity of this method is 10 copies/reaction. Compared with the sensitivity of the qPCR detection method recommended by the World Organization for Animal Health (WOAH, formerly OIE), the triple-RPA-LFS method established in this study is faster and simpler to operate. When applied to test 110 samples simultaneously with the laboratory standard testing method, the results of the qPCR detection matched the results of the laboratory standard method with a 100 % concordance rate. These experimental results indicate that the triple-RPA-LFS detection method established in this study has the characteristics of high specificity, high sensitivity, short detection time, and high accuracy. It can be used for rapid on-site detection and diagnosis of the three pathogens: WSSV, IHHNV, and TSV.
本研究聚焦于三种影响养殖虾的病毒，包括白斑综合征病毒（WSSV）、传染性皮下和造血组织坏死病毒（IHHNV）以及桃拉综合征病毒（TSV）。通过它们各自的保守基因片段设计特异性引物和探针，建立了一种同时检测WSSV、IHHNV和TSV的三重RPA-LFS检测方法。收集七种病原体和健康虾组织进行特异性试验。该方法能特异性扩增WSSV、IHHNV和TSV的基因片段，而从健康虾的肌肉组织或其他病原体中未扩增出片段，表明特异性强。对反应体系进行了优化，并验证了特异性和敏感性。采用连续稀释质粒法进行敏感性试验，确定该方法的检测灵敏度为10拷贝/反应。与世界动物卫生组织（WOAH，原OIE）推荐的qPCR检测方法的灵敏度相比，本研究建立的三重RPA-LFS方法操作更快、更简便。当与实验室标准检测方法同时应用于检测110个样本时，qPCR检测结果与实验室标准方法的结果匹配，符合率为100%。这些实验结果表明，本研究建立的三重RPA-LFS检测方法具有特异性高、灵敏度高、检测时间短和准确性高的特点。它可用于对WSSV、IHHNV和TSV这三种病原体进行快速现场检测和诊断。
