Direct-PCR检测柑橘黄龙病的快速制样方法研究

Research on DNA extraction methods for Direct-PCR detection of Citrus Huanglongbing

【目的】开发一种适用于Direct-PCR快速简便、有效的柑橘黄龙病病原DNA提取方法。【方法】从样品制备、提取速度以及DNA纯度等方面,分别比较研究了2种Direct-PCR制样方法与普通试剂盒制样方法提取柑橘黄龙病病原DNA的优缺点,并利用优化的制样技术分别比较了柑橘黄龙病病原16S rDNA的扩增效果。【结果】其中一种Direct-PCR制样方法所提取的柑橘叶片样品经RT-qPCR检测可以检测出黄龙病病原;通过优化制样后,该方法所提取的样品还可以通过常规PCR进行黄龙病病原检测。【结论】开发了一种更为快速简便、有效的柑橘黄龙病病原DNA提取方法,可为大批量快速检测柑橘黄龙病提供技术支撑。

【Objective】 To develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection. 【Method】 From aspects of preparing procedure, preparing time and the quality of DNA, advantages and disadvantages of three sample preparation methods were compared, including two Direct-PCR extraction methods and one general genomic DNA extraction kit method. In addition, PCR amplification effect of specific primers for 16S rDNA of Candidatus Liberibacter asiaticus have also been evaluated. 【Result】 The results showed that RT-qPCR could detect Ca. L. asiaticus by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method, 16S rDNA of Ca. L. asiaticus could also be amplified by routine PCR. 【Conclusion】 A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established, and provided technical supports for the preparation of large number of samples for detection of Ca. L. asiaticus.