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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45min,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。
For the prevention and control of tobacco bacterial wilt, rapid and efficient detection of pathogenic bacteria is necessary. Circular isothermal amplification (LAMP) is a novel method to rapidly complete target gene amplification under isothermal conditions. The FQY4 genomic sequence (NCBI number: CP004013) of the isolate from Guizhou isolates of Wilt disease was compared with the sequences of other published strains to identify the conserved region of the fliC gene encoding flagellin as a detection target. According to the principle of LAMP and the characteristics of the target sequence, a set of six loop-based isothermal amplification reaction system was designed. The results showed that incubation at 45 ℃ for 45 min could be used to detect single bacterial and pathogenic tobacco leaves of bacterial wilt and the results could be directly identified by naked eyes through color change or detected by agarose gel electrophoresis. By this new method, rapid identification of bacterial wilt colonies can be achieved, and it can also be used for rapid detection of suspected tobacco sick plants in the field.