番茄溃疡病菌的环介导等温核酸扩增技术

Detection of Clavibacter michiganensis subsp. Michiganensis by loop-mediated isothermal amplification

中文关键词:  番茄溃疡病菌  环介导等温核酸扩增技术  16SrRNA

英文关键词:Clavibacter michiganensis subsp. Michiganensis  LAMP  16SrRNA

中文摘要:

      目的 建立番茄溃疡病菌的环介导等温核酸扩增技术检测方法。方法 应用环介导等温核酸扩增技术(LAMP)对番茄溃疡病菌的16S rRNA特异性基因进行扩增, 采用4对引物识别目的片段的6个特异性区域, 62 ℃恒温1 h完成扩增。结果 设计的引物与对照菌皱纹假单胞菌、茄假单胞菌、丁香假单胞菌番茄致病变种都没有扩增反应, 表现了较好的特异性。这些对照菌在茄科蔬菜上易引起类似症状。结论 LAMP检测程序便捷, 所需设备简单, 其结果可以通过肉眼观察来判断, 比常规PCR灵敏且能更早得到反应结果, 因此该技术具有更大的优势。

英文摘要:

      Objective To establish a method for detection of Clavibacter michiganensis subsp. Michiganensis by loop-mediated isothermal amplification protocol (LAMP). Methods Detection of 16S rRNA of Clavibacter mi-chiganensis subsp. Michiganensis by a loop-mediated isothermal amplification protocol (LAMP) was evaluated in laboratory assays. LAMP amplified target DNA rapidly (1 h), isothermally (62 ℃) and with high-specificity based on four primers designed to recognize six independent sequences of target DNA. Results The LAMP primers did not react with suspensions of Pseudomonas corrugata, Pseudomonas solanacearum, and Pseudomonas syringae pv. tomato. The specificity of this LAMP assay is higher than PCR. Conclusion This method has convenient testing procedures, the required equipment is simple, and the results can be judged by the naked eye. It is sensitive than conventional PCR reaction and the results can be obtained earlier, so it has a greater advantage of the technology.