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园艺学报 ›› 2022, Vol. 49 ›› Issue (11): 2479-2488.doi: 10.16420/j.issn.0513-353x.2021-0606

• 新技术新方法 • 上一篇    下一篇

番茄枯萎病菌RT-PCR检测技术的建立与应用

孟臻1, 张伟萍1, 王莹1, 李龙3, 姬小雪1, 董贝2,**(), 乔康1,**()   

  1. 1山东农业大学植物保护学院,山东泰安 271018
    2济南市农业科学研究院,济南 250316
    3泰安市食品药品检验检测研究院,山东泰安 271000
  • 收稿日期:2022-06-24 修回日期:2022-08-16 出版日期:2022-11-25 发布日期:2022-11-25
  • 通讯作者: 董贝,乔康 E-mail:1985dongbei@163.com;qiaokang@sdau.edu.cn
  • 基金资助:
    山东省自然科学基金项目(ZR2021MC065);济南市农业科学院项目(yy201915)

Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f. sp. lycopersici

MENG Zhen1, ZHANG Weiping1, WANG Ying1, LI Long3, JI Xiaoxue1, DONG Bei2,**(), QIAO Kang1,**()   

  1. 1College of Plant Protection,Shandong Agricultural University,Tai’an,Shandong 271018,China
    2Jinan Academy of Agricultural Sciences,Jinan 250316,China
    3Tai’an Institute for Food and Drug Control,Tai’an,Shandong 271000,China
  • Received:2022-06-24 Revised:2022-08-16 Online:2022-11-25 Published:2022-11-25
  • Contact: DONG Bei,QIAO Kang E-mail:1985dongbei@163.com;qiaokang@sdau.edu.cn

摘要:

根据番茄枯萎病病原菌尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,FOL)蛋白激酶基因序列,设计1对番茄枯萎病特异性引物209F/361R,构建番茄枯萎病菌的RT-PCR(real-time PCR)检测体系,并利用该体系定量检测盆栽番茄茎基部病原菌。RT-PCR结果表明该引物只对番茄枯萎病菌有唯一产物吸收峰,对其他供试菌株都未检到荧光信号。利用该引物建立的RT-PCR检测体系线性关系良好,灵敏度为5.76 × 103 copies · μL-1,是普通PCR的10倍。人工接种番茄枯萎病菌定植后7 d即可在茎基部检测到病原菌;田间采集的24个自然发病样本中有16个能够检测到番茄枯萎病菌。基于RT-PCR技术构建的番茄枯萎病菌快速检测方法检测速度快、灵敏度高、特异性强、重现性好,能够为该病的早期诊断和流行监测提供理论依据。

关键词: 番茄枯萎病, 尖孢镰刀菌番茄专化型, 实时荧光定量PCR, 早期检测技术

Abstract:

In this study,a pair of specific primers,209F/361R,were designed to establish a real-time PCR(RT-PCR)reaction system of Fusarium oxysporum f. sp. lycopersici(FOL)based on the protein kinase gene sequences of FOL. FOL was detected at the base of tomato stems using this assay. Results of RT-PCR assays showed that only one absorption peak was detected with the primers for F. oxysporum and no absorption peak was produced for other tested strains. The RT-PCR detection system established with the primers showed a good linear relationship. The detection sensitivity was 5.76 × 103 copies · μL-1,which was 10 times more sensitive than the conventional PCR. Moreover,FOL was detected at the base of the tomato stems at the 7th day after artificial inoculation. Twenty-four naturally infected tomato samples were collected from field condition and the RT-PCR results showed that FOL was detected in 16 samples. The established RT-PCR detection system for F. oxysporum was fast,highly specific,sensitive,and reproducible,providing scientific tools for epidemic monitoring and early diagnosis of Fusarium wilt of tomato.

Key words: Tomato Fusarium wilt, Fusarium oxysporum f. sp. lycopersici, RT-PCR, early detection technology

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