浙江农业学报 ›› 2017, Vol. 29 ›› Issue (1): 113-118.DOI: 10.3969/j.issn.1004-1524.2017.01.16

• 植物保护 • 上一篇    下一篇

柑橘溃疡病菌环介导等温扩增快速检测技术研究

顾渊1, 岑铭松1, 2, 马海杰1, 李红叶1, *   

  1. 1.浙江大学 农业与生物技术学院 生物技术研究所,浙江 杭州 310058;
    2.杭州市植保土肥总站, 浙江 杭州 310004
  • 收稿日期:2016-06-22 出版日期:2017-01-15 发布日期:2017-02-23
  • 通讯作者: 李红叶, E-mail:hyli@zju.edu.cn
  • 作者简介:顾渊(1993—),男,浙江嘉兴人,主要从事植物病理学研究。E-mail:3120100135@zju.edu.cn
  • 基金资助:
    杭州市农业科研攻关项目(20150432B14)

Rapid detection of citrus bacterial canker by loop-mediated isothermal amplification

GU Yuan1, CEN Mingsong1, 2, MA Haijie1, LI Hongye1, *   

  1. 1. Institute of Biotechnology,College of Agriculture and Biotechnology,Zhejiang University, Hangzhou 310058, China;
    2. Hangzhou Plant Protection and Soil-fertilizer Station, Hangzhou 310004, China
  • Received:2016-06-22 Online:2017-01-15 Published:2017-02-23

摘要: 环介导等温扩增(loop-mediated isothermal amplification, LAMP)快速检测已用于基层植物检验检疫中的疑似样品检测诊断。柑橘溃疡病菌(Xanthomonas citri subsp. citri)是重要的检疫性病害,根据柑橘溃疡病菌基因组中一段保守序列设计LAMP引物,建立了柑橘溃疡病菌的LAMP检测方法。该LAMP检测方法只对柑橘溃疡病菌DNA和柑橘溃疡病病斑总DNA进行扩增,而对非靶标菌DNA和非靶标病斑总DNA不发生扩增,特异性与PCR一致。该LAMP检测方法最低可检出100 pg的纯柑橘溃疡病菌DNA和最低每微升4个细菌,灵敏度均比PCR高了1 250倍。LAMP检测法能很好地区分症状极易混淆的柑橘溃疡病和疮痂病。该试验建立的柑橘溃疡病菌LAMP检测方法具有简单、快速、灵敏和特异等优点,可在条件相对简陋的实验室应用。

关键词: 柑橘溃疡病, 病害检测, 环介导等温扩增(LAMP), 常规PCR

Abstract: Citrus canker (Xanthomonas citri subsp. citri) is an important quarantine disease. Previous studies showed that loop-mediated isothermal amplification (LAMP) can be used for rapid detection of suspected citrus canker samples. Based on a new conserved genome DNA sequence, we designed LAMP primers and established a LAMP detection method for detection of citrus canker samples in this study. The specificity of established LAMP detection method was consistent with that of regular PCR. However, this LAMP detection method could detect as low as 100 pg DNA of X. citri subsp. citri, and only 4 cells per microliter of X. citri, sensitivity of LAMP was 1 250 times higher than that of the regular PCR. The LAMP assay could distinguish citrus canker from citrus scab, the two diseases easily confused based on symptoms. Therefore, we suggest that LAMP detection method for citrus canker established in this study is simple, rapid, sensitive and specific, it could be used in laboratory with relatively simple conditions.

Key words: citrus canker, Xanthomonas citri subsp. citri, detection, loop-mediated isothermal amplification (LAMP), regular PCR

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