Abstract
Rice stripe virus (RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies (MAbs) 16E6 and 11C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper (SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20480 (w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560 (individual SPBH/µL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.
概 要
目 的
制备一种检测水稻植物和传毒介体灰飞虱中水稻条纹病毒 (RSV) 的胶体金免疫层析试纸条, 为水稻条纹叶枯病的田间调查和检测以及预测预警提供快速、实用的检测试剂。
创新点
利用制备的 RSV 单克隆抗体, 首次制备了能在 5~10 min 内快速、准确、灵敏、特异地检测水稻植物和灰飞虱体内 RSV 的胶体金免疫层析试纸条。
方 法
以差速离心方法提纯的 RSV 病毒粒子作为免疫原免疫 BALB/c 小鼠, 通过杂交瘤技术获得了高度特异和灵敏的RSV 单克隆抗体。采用柠檬酸钠还原氯金酸的方法制备胶体金并标记一个 RSV 单克隆抗体, 另一个 RSV 单克隆抗体和羊抗鼠抗体分别包被到硝酸纤维素膜的检测线和质控线, 将吸水滤纸制成的样品垫、RSV 免疫胶体金垫、结合有 RSV 单抗和羊抗鼠抗体的硝酸纤维素膜和 吸水纸依次粘贴到聚氯乙烯 (PVC) 胶板上研制检测水稻植物和传毒介体灰飞虱中 RSV 的胶体金免疫层析试纸条。对田间样品进行 RSV 检测, 分析试纸条检测 RSV 的有效性。
结 论
利用杂交瘤技术获得了2 株 RSV 单克隆抗体 (16E6 和11C1), 胶体金标记 16E6 单克隆抗体 包被在聚酯膜制成的结合垫上, 以 11C1 单克隆 抗体和羊抗鼠抗体分别包被到硝酸纤维素膜的检测线和质控线, 制成能在5~10 min 内快速、特异、灵敏、准确地检测田间水稻及单头灰飞虱样品中 RSV 的胶体金免疫层析试纸条。试纸条检测感染 RSV 的水稻植株和灰飞虱呈阳性反应, 而检测健康水稻和感染其它5 种常见水稻病毒的植株及其传毒介体呈阴性反应, 且试纸条检测 RSV 感染水稻植物组织的灵敏度达到 1:20 480 倍稀释 (w/v, g/mL), 检测携带 RSV 单头灰飞虱的灵敏度达到1:2560 倍稀释 (单头灰飞虱/μL)。田间样品检测结果发现, 试纸条检测结果与反转录聚合酶链反应 (RT-PCR) 的检测结果一致, 表明制备的试纸条可有效地用于田间 RSV 的检测, 从而为水稻条纹叶枯病的诊断、监测预警、抗病育种、流行病学研究及科学防治提供技术和物质支撑。
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Acknowledgements
We are grateful to Dr. ** Zhou & Jian-xiang Wu
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Project supported by the National Key Research and Development Program of China (No. 2016YFD0300706), the Ministry of Agriculture of China (No. 2016ZX08009003-001), the National Natural Science Foundation of China (No. 31571976), and the Earmarked Fund for China Agriculture Research System (No. nycytx-001)
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Huang, Dq., Chen, R., Wang, Yq. et al. Development of a colloidal gold-based immunochromatographic strip for rapid detection of Rice stripe virus. J. Zhejiang Univ. Sci. B 20, 343–354 (2019). https://doi.org/10.1631/jzus.B1800563
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DOI: https://doi.org/10.1631/jzus.B1800563
Key words
- Monoclonal antibody
- Colloidal gold-based immunochromatographic strip
- Rice stripe virus
- Rice
- Small brown planthopper