草鱼GCRV-II抗原检测胶体金试剂盒研发与应用
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S 941.41+1

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农业生物育种国家科技重大专项(2023ZD04065)


Development and application of a colloidal gold detection kit for rapid detection of type II grass carp reovirus
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    摘要:

    目的 基于胶体金免疫层析方法研发草鱼呼肠孤病毒II型 (GCRV-II) 快速检测技术。方法 利用特异识别GCRV-II外衣壳蛋白VP35的两株不同抗原表位单克隆抗体VP35#8和VP35#18,采用双抗体夹心法研发速测技术,通过20 nm胶体金颗粒标记VP35#8抗体制备金标垫反应区,VP35#18抗体包被于硝酸纤维素膜 (NC膜) ,T线作为检测区, C线标记羊抗鼠IgG作为质控区,各组分依次组装获得GCRV-II胶体金免疫层析试纸条用于制备检测试剂盒。结果 该试剂盒在10 min内可完成样本检测,且对鲤春病毒血症病毒 (SVCV) 、鲤疱疹病毒III型 (CyHV-3) 及草鱼GCRV-I均无交叉反应。使用本试剂盒随机检测10尾草鱼样本及感染GCRV-II稀有鮈鲫的不同组织,其检测结果与GCRV-II国标法 (GB/T 36190—2018) 检测结果相符,进一步证实本试剂盒具有较好的检测准确性。灵敏度验证实验和稳定性验证实验结果显示,试剂盒检测灵敏度高达1.76×102 拷贝/μL,4~25 ℃储存12个月后检测线与质控线仍正常显色,储存稳定性良好。结论 本研究成功研制出GCRV-II抗原检测试剂盒,且该试剂盒具备操作便捷、快速及高特异性等优点,对GCRV-II快速诊断和草鱼出血病防控具有重要实践意义。本研究将进一步推动水生病毒性疾病快速检测技术的发展,从而为我国鱼类健康养殖和病毒性疾病的防控提供关键技术支撑。

    Abstract:

    Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus type II (GCRV-II), is the most devastating viral disease affecting grass carp aquaculture in China. GCRV-II is highly virulent and can rapidly spread, leading to substantial economic losses annually. Current detection of GCRV still heavily relies on laboratory-based molecular techniques, which are complex and fail to meet the urgent need for on-site rapid diagnosis. To address this, this study aims to develop a rapid detection kit for GCRV-II based on colloidal gold immunochromatography. Two monoclonal antibodies, VP35#8 and VP35#18, specifically recognizing two distinct epitopes of the outer capsid protein VP35 of GCRV-II, were utilized. The VP35#8 antibody was conjugated with 20 nm colloidal gold particles to prepare the gold-labeled pad, while the VP35#18 antibody and goat anti-mouse IgG were immobilized on the test line (T-line) and control line (C-line) of the nitrocellulose membrane, respectively, to assemble the test strip. The results demonstrated that the kit could successfully detect GCRV-II from diseased C. idella and Gobiocypris rarus within 10 minutes. It showed excellent specificity with no cross-reactivity against Spring Viraemia of Carp Virus (SVCV), Cyprinid Herpesvirus 3 (CyHV-3), and GCRV-I. The detection sensitivity reached 1.76×102 copies/μL, and the kit remained stable after storage at 4–25°C for 12 months. The results were consistent with that obtained by the national standard method (GB/T 36190-2018). In summary, this study successfully developed a user-friendly, rapid, and reliable on-site detection tool for GCRV-II, providing solid technical support for the early diagnosis and control of C. idella hemorrhagic disease, which is of great significance for safeguarding the sustainability of the aquaculture industry.

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吴海霞,冯建华,芦雅南,高娃,闫慧,许丹,计晓花,吕利群,徐镇,王俊亚,邹钧.草鱼GCRV-II抗原检测胶体金试剂盒研发与应用[J].水产学报,2026,50(3):039414

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  • 收稿日期:2025-07-30
  • 最后修改日期:2025-09-10
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  • 在线发布日期: 2026-01-24
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