| 苏怡敏 1, 朱鹏飞 3, 钟鑫强 1, 王 深 4, 张博轩 1, 张春雨 5, 李小宇 5, 王永志 2*.葱潜隐病毒序列分析、单克隆抗体制备及ELISA检测方法建立[J].植物保护,2026,52(4):301-309. |
| 葱潜隐病毒序列分析、单克隆抗体制备及ELISA检测方法建立 |
| Sequence analysis, monoclonal antibody preparation and ELISA development for shallot latent virus |
| 投稿时间：2025-09-16 修订日期：2025-11-20 |
| DOI：10.16688/j.zwbh.2025474 |
| 中文关键词: 分蘖洋葱 葱潜隐病毒 单克隆抗体 ELISA 序列分析 |
| 英文关键词:shallot shallot latent virus monoclonal antibody ELISA sequence analysis |
| 基金项目:吉林省农业科技创新工程(CXGC2024RCY019) |
| 作者 | 单位 | E-mail | | 苏怡敏 1, 朱鹏飞 3, 钟鑫强 1, 王 深 4, 张博轩 1, 张春雨 5, 李小宇 5, 王永志 2* | 1. 吉林农业大学植物保护学院, 长春 130118
2. 吉林农业大学生命科学学院, 长春 130118
3. 吉林农业大学动物科学技术学院, 长春 130118
4. 吉林农业大学农学院, 长春 130118
5. 吉林省农业科学院(中国农业科技东北创新中心)植物保护研究所, 公主岭 136100 | yzwang@126.com |
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| 中文摘要: |
| 葱潜隐病毒(shallot latent virus, SLV)是严重危害葱属作物产量和品质的重要病原。近年来, 在珠葱优势产区, SLV隐性感染频发, 并常与其他病毒复合侵染, 严重威胁产业健康发展。为实现该病原的早期诊断与有效防控, 本研究系统分析了吉林分离物SLV与大蒜潜隐病毒(garlic latent virus, GLV)的CP基因。SLV 与 GLV 的 CP 基因核苷酸一致性为 77.3%~79.9%, 氨基酸一致性为 93.7%~97.2%, 均高于 Carlavirus 病毒种划分的阈值, 支持二者属于同一病毒种。基于CP蛋白的保守性, 以原核表达的SLV CP蛋白为抗原, 免疫小鼠并获得3株特异性单克隆抗体(4E9、5B11和8F11)。经配对筛选, 确定以5B11为捕获抗体、HRP标记的5B11为检测抗体, 建立了双抗体夹心ELISA(enzyme-linked immunosorbent assay)体系。该体系可在1 h内完成检测。灵敏度检测显示, 感病叶片提取液80倍稀释, 对洋葱黄矮病毒(onion yellow dwarf virus, OYDV)、青葱X病毒(shallot virus X, SVX)等无交叉反应。在40份田间样本中, 其检测结果与RT-PCR方法一致率为92.5%。系统发育分析显示, 吉林地区 SLV 群体由本地优势株系、区域内混合株系及可能的外源相关株系组成, 反映出其在区域尺度上具有一定的遗传复杂性。本研究所得结果为 SLV 的快速检测及健康种球筛选提供了方法支持, 同时为 SLV 与GLV 的分类确认及其在该地区的种群结构特征提供了分子依据。 |
| 英文摘要: |
| Shallot latent virus (SLV) is an important pathogen that severely affects the yield and quality of Allium crops. In recent years, latent infection by SLV has frequently occurred in major shallot-producing regions and is often associated with mixed infections by other viruses, posing a serious threat to the sustainable development of the industry. To enable early diagnosis and effective control of this pathogen, the coat protein (CP) genes of SLV and garlic latent virus (GLV) isolates from Jilin province were systematically analyzed. The nucleotide identity of the CP genes between SLV and GLV ranged from 77.3% to 79.9%, and the amino acid identity ranged from 93.7% to 97.2%, both exceeding the species demarcation threshold of the genus Carlavirus, supporting their classification as the same viral species. Based on the conserved CP protein, a prokaryotically expressed SLV CP protein was used as an antigen to immunize mice, and three specific monoclonal antibodies (4E9, 5B11, and 8F11) were obtained. Following paired screening, 5B11 was selected as both the capture antibody and the HRP-labeled detection antibody to establish a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA). The assay can be completed within 1 h. Sensitivity testing showed that an 80-fold dilution of infected leaf extracts exhibited no cross-reactivity with onion yellow dwarf virus (OYDV), shallot virus X (SVX), or other tested viruses. In 40 field samples, the detection results showed a 92.5% consistency with RT-PCR. Phylogenetic analysis revealed that the SLV population in Jilin consists of locally dominant strains, regionally mixed strains, and potentially exogenous related strains, reflecting a certain degree of genetic complexity at the regional scale. These results provide methodological support for the rapid detection of SLV and the screening of healthy seed bulbs, and offer molecular evidence for the taxonomic classification of SLV and GLV as well as their population structure characteristics in this region. |
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