| 董经晗 1, 2, 李 鑫 2, 巴哈提古丽·马那提拜 2, 王 翀 2*, 李 杨 3*, 古扎丽阿依·牙生 2, 罗 明 1, 高晓珂 1, 2, 田沁怡 1, 2.瓜蔓枯病菌RPA/CRISPR-Cas12a快速可视化检测方法的建立[J].植物保护,2026,52(4):293-300. |
| 瓜蔓枯病菌RPA/CRISPR-Cas12a快速可视化检测方法的建立 |
| Establishment of a rapid visual detection method for gummy stem blight pathogen based on RPA/CRISPR-Cas12a |
| 投稿时间：2025-09-17 修订日期：2025-11-26 |
| DOI：10.16688/j.zwbh.2025476 |
| 中文关键词: 瓜蔓枯病 重组酶聚合酶扩增(RPA) CRISPR-Cas12a 快速检测 可视化检测 |
| 英文关键词:gummy stem blight of melons recombinase polymerase amplification (RPA) CRISPR-Cas12a rapid detection visual detection |
| 基金项目:海关总署科研项目(2023HK017);新疆维吾尔自治区自然科学基金(2023D01A67) |
| 作者 | 单位 | E-mail | | 董经晗 1, 2, 李 鑫 2, 巴哈提古丽·马那提拜 2, 王 翀 2*, 李 杨 3*, 古扎丽阿依·牙生 2, 罗 明 1, 高晓珂 1, 2, 田沁怡 1, 2 | 1. 新疆农业大学农学院, 乌鲁木齐 830052
2. 乌鲁木齐海关技术中心, 乌鲁木齐 830013
3. 石河子大学生命科学学院, 石河子 832000 | 王翀wchgq@126.com;李杨502193182@qq.com |
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| 中文摘要: |
| 瓜蔓枯病菌Stagonosporopsis cucurbitacearum主要侵染茎蔓、叶片及果实, 严重时造成全株枯死。该病菌是出口瓜类种子中的主要检疫对象, 面对出口产品检疫要求的不断提升, 亟须对检测技术进行升级和创新。本研究针对瓜蔓枯病菌的dockerin-like protein保守基因, 设计出特异性的 RPA 引物及 Cas12a crRNA, 通过试验确定荧光法中报告分子浓度为500 nmol/L, 试纸条法中报告分子浓度为250 nmol/L, 建立了基于RPA/CRISPR-Cas12a 系统的瓜蔓枯病菌可视化检测方法。RPA/CRISPR-Cas12a检测方法从样品处理、RPA扩增到CRISPR反应整个过程可在70 min内完成, 检测灵敏度为33 fg/μL。该方法对不同菌株均显示出良好的特异性。模拟带菌检测试验表明该方法与 qPCR 检测结果一致。本研究为瓜类种子在出口贸易中快速通关提供有力技术支撑, 同时也适用于瓜类蔓枯病在田间的早期诊断、监测和预警。 |
| 英文摘要: |
| Stagonosporopsis cucurbitacearum mainly infects stems, leaves and fruits, and can cause whole-plant death in severe cases. This pathogen is a key quarantine item for exported melon seeds. Facing the continuous improvement of quarantine requirements for export products, there is an urgent need to upgrade and innovate detection technologies. In this study, specific RPA primers and Cas12a crRNA were designed targeting the dockerin-like protein conserved gene of S.cucurbitacearum. A visual detection method for S.cucurbitacearum based on the RPA/CRISPR-Cas12a system was established with the optimal reporter concentration of 500 nmol/L for fluorescence detection and 250 nmol/L for lateral flow strip detection. The RPA/CRISPR-Cas12a detection method can be completed within 70 minutes from sample processing and RPA amplification to CRISPR reaction, with a detection sensitivity of 33 fg/μL. This method showed good specificity to different strains. Simulated infected sample detection experiments showed that the results were consistent with those of qPCR. This study provides strong technical support for the rapid customs clearance of melon seeds in export trade, and is also suitable for the early diagnosis, monitoring, and early warning of melon gummy stem blight in the field. |
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