| 摘要: |
| 针对烟草青枯病的防控，开发快速、高效的病原菌检测方法是必要的。环式等温扩增（LAMP）是一种在等温条件下快速完成靶标基因扩增的新方法。将青枯病菌贵州分离株FQY4基因组序列（NCBI号：CP004013）与其他已发表菌株序列比对分析，确定编码鞭毛蛋白的fliC基因的保守区域可被选作检测靶标。根据LAMP原理以及靶标序列的特点，设计了一组由6条引物组成的环式等温扩增反应体系。结果表明：61 ℃条件下孵育45 min，可完成对青枯病病原菌单菌落和带菌烟草叶片的检测，结果判定可以通过颜色变化被肉眼直接识别，也可以通过琼脂糖凝胶电泳检测。通过这种新方法，可以实现对青枯病菌菌落的快速鉴别，还可以用于对田间疑似带病烟草植株的快速检测。 |
| 关键词: 烟草 茄科劳尔氏菌 fliC基因 环介导的等温扩增 |
| DOI：10.11841/j.issn.1007-4333.2014.01.13 |
| 投稿时间：2013-07-23 |
| 基金项目:贵州省科学技术基金项目（黔科合J字[2013]2194号）；中国烟草总公司贵州省公司科技计划项目（201022） |
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| Rapid and sensitive detection method for Ralstonia solanacearum based on Loop-mediated isothermal amplification |
|
JIA Meng-ao1, CHEN Xing-jiang1, LIN Ye-chun1,2, SHANG Sheng-hua1
|
| (1.Key Laboratory of Molecular Genetics, Guizhou Academy of Tobacco Science, Guiyang 550081, China;2.College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| Tobacco bacterial wilt disease,caused by Ralstonia solanacearum,is one of the most important biotic stresses for tobacco production in Guizhou province,southwest China.It is necessary to develop a rapid and sensitive pathogen detection method for wilt disease management.Here we reported a new detection strategy based on Loop-mediated isothermal amplification (LAMP) for Ralstonia solanacearum diagnosis.Referring to the genome of Ralstonia solanacearum FQY4 (NCBI accession No.CP004013),which is the dominant strain in Guizhou province,aligned with the published sequences of other strains,the fliC gene which encoded the flagellar protein,was chosen as the detection target.We designed a set of 6 LAMP primers based on the sequence of the conserved region of fliC gene,and optimized the reaction condition as 60 min at 60 ℃.The amplification result,which reflected whether the pathogen existed or not,could be visual detected by colors identification,or could be presented as ladder-like bands pattern by the DNA product electrophoresis in agarose gel.Using this method,not only the Ralstonia solanacearum isolated colony but also the tobacco plant carrying pathogen could be identified rapidly and accurately. |
| Key words: tobacco bacterial wilt disease Ralstonia solanacearum fliC gene Loop-mediated isothermal amplification |
