| 阿依加玛丽·阿卜杜扎伊尔 1, 2, 魏浩宇 1, 3, 古丽努尔·艾合买提 1, 2, 王梦茜 1, 韩 盛 4, 赵廷昌 1, 关 巍 1, 杨玉文 1*, 麦合木提江·米吉提 2*.瓜类细菌性果斑病LAMP-CRISPR/Cas12a检测技术体系的建立及应用[J].植物保护,2026,52(4):310-321. |
| 瓜类细菌性果斑病LAMP-CRISPR/Cas12a检测技术体系的建立及应用 |
| Development and application of a loop-mediated isothermal amplification coupled with CRISPR/Cas12a detection system for bacterial fruit blotch |
| 投稿时间：2025-10-16 修订日期：2026-01-02 |
| DOI：10.16688/j.zwbh.2025533 |
| 中文关键词: 瓜类细菌性果斑病 CRISPR 环介导等温扩增 检测技术 |
| 英文关键词:bacterial fruit blotch CRISPR loop-mediated isothermal amplification. |
| 基金项目:国家重点研发计划(2023YFD1401200)；新疆维吾尔自治区自然科学基金(2022D01A271)；新疆维吾尔自治区重大专项(2023A02009)；国家西甜瓜产业技术体系 (CARS-25) |
| 作者 | 单位 | E-mail | | 阿依加玛丽·阿卜杜扎伊尔 1, 2, 魏浩宇 1, 3, 古丽努尔·艾合买提 1, 2, 王梦茜 1, 韩 盛 4, 赵廷昌 1, 关 巍 1, 杨玉文 1*, 麦合木提江·米吉提 2* | 1. 中国农业科学院植物保护研究所, 植物病虫害综合治理全国重点实验室, 北京 100193
2. 新疆农业大学农学院, 农业农村部西北荒漠绿洲农林外来入侵生物防控重点实验室(部省共建), 乌鲁木齐 830052
3. 沈阳农业大学植物保护学院, 沈阳 110161
4. 新疆维吾尔自治区农业科学院植物保护研究所, 乌鲁木齐 830091 | 杨玉文yangyuwen@caas.cn;麦合木提江·米吉提287600102@qq.com |
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| 中文摘要: |
| 瓜类细菌性果斑病(bacterial fruit blotch)是由西瓜副噬酸菌Paracidovorax citrulli引起的严重危害葫芦科作物的一种世界性病害, 对我国西瓜甜瓜产业造成重大的损失。建立高通量、灵敏度高、特异性强且便于操作的瓜类种子携带细菌性果斑病的检测方法, 对保障种子健康和控制苗期病害具有重要意义。本研究设计并筛选出检测西瓜副噬酸菌的LAMP与CRISPR特异性引物, 优化了 LAMP-CRISPR/Cas12a 检测体系中各成分最佳配比浓度, 进行了引物特异性、灵敏度、重复性检测。结果表明, 在 65℃ 恒温条件下, LAMP反应10 min, CRISPR/Cas12a 39℃恒温反应10 min, 仅需20 min即可完成瓜类细菌性果斑病LAMP-CRISPR/Cas12a检测, 该方法特异性高, 最低检测限为1 拷贝/μL, 且具有良好的重复性(批内变异系数<5%, 批间变异系数<15%)。甜瓜组织的检测结果显示, 该体系可从种子、发病植株组织等样本中检测到目标病原菌, 且种子带菌检测可以检测到500 粒健康种子中的1 粒带菌种子, 实现种子带菌的高通量检测, 且检测结果可视化。该体系的建立与应用将为瓜类细菌性果斑病的田间实时快速检测提供一种高效的方法。 |
| 英文摘要: |
| Bacterial fruit blotch, caused by Paracidovorax citrulli, is a worldwide devastating disease that severely affects cucurbit crops, inflicting significant losses on watermelon and melon industries in China. Establishing a high-throughput, highly sensitive, specific, and user-friendly detection method for P.citrulli in cucurbit seeds is of great significance for ensuring seed health and controlling seedling-stage diseases. In this study, specific LAMP and CRISPR primers for P.citrulli were designed and screened. The optimal concentration ratios of components in the LAMP-CRISPR/Cas12a detection system were optimized, followed by assessments of primer specificity, sensitivity, and repeatability. The results demonstrated that the LAMP-CRISPR/Cas12a assay for detection of bacterial fruit blotch could be completed within only 20 minutes, consisting of a 10-minute LAMP reaction at a constant temperature of 65℃ and a 10-minute CRISPR/Cas12a reaction at a constant temperature of 39℃. This method exhibited high specificity with a minimum detection limit of one copy/μL. It also showed good repeatability, with intra-assay coefficient of variation <5% and inter-assay coefficient of variation <15%. Detection results from melon tissues indicated that the system could detect P.citrulli from samples such as seeds and diseased plant tissues. Notably, in seed-borne pathogen detection, it could identify one infected seed among 500 healthy seeds, enabling high-throughput detection of seed-borne pathogens with visualizable results. The establishment and application of this system provide an efficient method for real-time and rapid field detection of bacterial fruit blotch. |
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