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中国农学通报

中国农学通报 ›› 2025, Vol. 41 ›› Issue (31): 102-110.doi: 10.11924/j.issn.1000-6850.casb2025-0065

• 植物保护·农药 • 上一篇    下一篇

荷花腐败病菌的实时荧光LAMP快速检测技术的建立及应用

曾莉莎1(), 温华强2, 麦进培1, 李燕荷1, 周海琪1, 郑芝波1(), 吕顺1, 周而勋2   

  1. 1 东莞市农业科学研究中心，广东东莞 523000
    2 华南农业大学植物保护学院/广东省微生物信号与作物病害防控重点实验室，广州 510642
  • 收稿日期:2025-02-06 修回日期:2025-08-15 出版日期:2025-11-05 发布日期:2025-11-07
  • 通讯作者:
    郑芝波，女，1973年出生，福建漳平人，研究员，硕士，研究方向：花卉栽培利用。通信地址：523086 东莞市南城区绿色路市现代农业科技园东莞市农业科学研究中心，Tel：0769-23668938，E-mail：。
  • 作者简介:

    曾莉莎，女，1985年出生，广东海丰人，研究员，硕士，研究方向：植物病理学。通信地址：523086 东莞市南城区绿色路市现代农业科技园东莞市农业科学研究中心，Tel：0769-23668928，E-mail：。

  • 基金资助:
    广东省科技计划项目“荷花腐败病抗病品种筛选及利用”(2015A020209002); 东莞市社会科技发展重点项目“荷花腐败病发病机理与生态防控技术研究”(2014108101044)

Establishment and Application of Real-time Fluorescent LAMP Rapid Detection of Lotus Rhizome Rot Pathogen

ZENG Lisha1(), WEN Huaqiang2, MAI Jinpei1, LI Yanhe1, ZHOU Haiqi1, ZHENG Zhibo1(), LYU Shun1, ZHOU Erxun2   

  1. 1 Dongguan Agricultural Research Centre, Dongguan, Guangdong 523000
    2 College of Plant Protection, South China Agricultural University/ Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Guangzhou 510642
  • Received:2025-02-06 Revised:2025-08-15 Published:2025-11-05 Online:2025-11-07

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摘要：

为构建一种高效、灵敏的荷花腐败病菌(Fusarium commune)实时荧光环介导等温扩增技术LAMP(Real-time fluorescent LAMP)检测体系，以荷花腐败病菌的翻译延伸因子(TEF-1α)序列作为检测靶标，设计并筛选出4条特异性LAMP引物，成功建立了基于实时荧光监测的LAMP快速检测方法。系统性验证实验表明，该方法具备显著优势，在63℃恒温条件下，60 min即可完成病原菌检测，且具有高度特异性，能精准识别荷花腐败病菌。针对供试菌株的测试结果显示，10株荷花腐败病菌反应结果呈绿色（阳性），电泳结果出现梯度条带，实时荧光扩增正常且熔解曲线为单一尖峰；而其他5株对照菌株反应结果呈橙色（阴性），电泳无梯度条带，实时荧光无扩增。灵敏度实验表明，利用LAMP技术检测F. commune的最低检测限为100 fg/μL，土壤中分生孢子的最低检测限为1×103个/(g·土壤)，制作的标准曲线可用于F. commune的定量检测。本方法适用于田间植株受F. commune侵染前后的检测，其地下茎组织F. commune的DNA含量与其发病程度呈正相关。本研究建立的荷花腐败病菌实时荧光LAMP快速检测方法具有较高的特异性和灵敏度，操作简便快捷，适用于F. commune的田间快速检测。

关键词: 荷花腐败病菌, TEF-1α基因序列, 实时荧光, 环介导等温扩增, 快速检测, 分子检测, 特异性引物

Abstract:

In order to develop a rapid and sensitive real-time fluorescent LAMP technique for quantitative detection of lotus rhizome rot pathogen (Fusarium commune), four LAMP specific primers were designed based on TEF-1α gene sequence unique to F. commune, a real-time fluorescent LAMP rapid detection method for pathogen of lotus rhizome rot was established, and the specificity, sensitivity and field application effects of this method were evaluated. The specific test showed that this method could identify and detect the lotus rhizome rot pathogen (F. commune) in 60 minutes under the constant temperature of 63℃. Among the 15 tested strains, 10 strains showed positive green and gradient bands in the electrophoretic test results, while the other 5 control strains showed orange and no gradient bands. The results of real-time fluorescent LAMP showed that the F. commune strains could expand normally with a single peak dissolution curve, while other control strains did not expand. Sensitivity experiments showed that the minimum detection limit of LAMP technology for F. commune was up to 100 fg/μL, and the minimum limit of soil strain content was 1×103 spores per gram of soil. The standard curve of pathogen developed in this paper can be used for the quantitative detection of F. commune. This technique was also suitable for the detection of F. commune in lotus plants in the field before and after infection. The DNA content of F. commune in the underground rhizome tissue of lotus plants was positively correlated with the severity of the disease. The real-time fluorescent LAMP rapid detection technology established in this paper has the advantages of strong specificity, high sensitivity, simple and quick operation, which is suitable for the field rapid detection of F. commune.

Key words: lotus rhizome rots pathogen, TEF-1α gene sequence, real-time fluorescent, loop-mediated isothermal amplification, rapid detection, molecular testing, specific primers