基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌

发布时间：2018-03-12 13:40

本文选题：番茄溃疡病菌　切入点：环介导等温扩增　出处：《植物病理学报》2017年05期 　论文类型：期刊论文

【摘要】：本研究选取番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)致病岛上的chpC基因的部分序列,作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)靶标片段进行LAMP引物设计。对反应体系优化后进行特异性测定,结果表明供试的89株番茄溃疡病菌中86株检测结果为阳性,3株为阴性,供试的14株非番茄溃疡病菌(其他重要植物病原细菌)均为阴性。检测番茄溃疡病菌菌悬液样品的阈值为4.8×10~5 CFU·mL~(-1),对DNA样品的检测阈值为1.8×10~(-2) ng·μL~(-1),并据此建立了番茄溃疡病菌的LAMP检测方法。将该方法应用于番茄种子携带Cmm的检测,通过提取种子浸提液样品的总DNA,实现了对番茄种子携带Cmm的直接检测。与普通PCR相比,该方法更加快捷简便,不依赖PCR仪等昂贵的仪器设备,可以丰富现有的番茄溃疡病菌分子检测体系,为口岸等检疫部门提供简单易行的检测初筛手段。
[Abstract]:This study selected tomato Canker (Clavibacter michiganensis subsp.michiganensis, Cmm) partial sequence of chpC gene of pathogenic Island, as a loop mediated isothermal amplification (loop-mediated isothermal, amplification, LAMP) target fragments were LAMP primers were designed. The optimization of the reaction system after the specific determination, the results showed that 86 positive strains detected in 89 strains the tomato canker pathogen tested, 3 strains were negative, 14 strains of non tomato canker pathogen tested (other important plant pathogenic bacteria) were negative. Detection of tomato canker bacterium suspension sample threshold is 4.8 * 10~5 CFU mL~ (-1), the detection threshold of DNA sample is 1.8 * 10~ (-2) ng - L~ (-1), and set up a LAMP method for detection of tomato canker. The method was applied to detect tomato seeds with Cmm, the total DNA extract samples by extracting seed leaching, the realization of a tomato The direct detection of Cmm is carried out. Compared with ordinary PCR, the method is more convenient and fast, and does not rely on expensive instruments such as PCR. It can enrich the existing molecular detection system of tomato canker, and provide a simple and feasible screening and screening method for port and other quarantine departments.

【作者单位】：中国农业大学植物病理学系种子病害检验与防控北京市重点实验室;
【基金】：国家自然科学基金项目(31571972)
【分类号】：S436.412.1

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