乌头霜霉病病原物分子检测方法的建立

目的:建立乌头霜霉病病原菌快速分子检测方法,为乌头种苗检疫及栽培环境土壤安全性评价提供依据。方法:使用真菌DNA提取试剂盒提取病原菌DNA,采用真菌核糖体内转录间隔区(ITS)通用引物ITS1/ITS4,对病原菌的r DNA-ITS区间序列进行扩增,将聚合酶链式反应(PCR)产物回收纯化并进行序列测定,并将测得的ITS序列同Gene Bank中搜索到的相关ITS序列进行比较,运用DNAMAN比对出特异序列片段,利用特异序列片段通过Primer Premier 5.0设计并筛选特异性引物,建立乌头霜霉病病原菌快速PCR检测方法。结果:从Primer Premier 5.0设计的8对引物中筛选出了灵敏度高、特异性强的引物对L1A/L1B,该引物能够从乌头霜霉病病原菌DNA扩增出670 bp的具有检测价值的明亮条带,利用该引物也能够检测出乌头种苗、成株以及栽培土壤是否存在乌头霜霉病原菌。结论:筛选出的引物对L1A/L1B能够快速、简便、有效地检测出乌头霜霉病病原菌,建立的方法能用于对乌头种苗霜霉病的提前检测或检疫。

Establishment of Molecular Detection Method for Pathogen of Aconiti Downy Mildew

Objective: To establish the rapid molecular detection method for pathogen of aconiti downy mildew and provide the basis for the quarantine of the A. carmichaeli seedling and safety evaluation of the cultivated environmental land. Method: Pathogen DNA was extracted by fungal DNA kit and rDNA-ITS sequence of pathogen was amplified by ITS1/ITS4 universal primers of ribosomal internal transcribed spacer (ITS) in fungi; the polymerase chain reaction (PCR) product was recycled, purified and sequenced. The measured ITS sequences were then compared with the related ITS sequences searched in GeneBank; specific sequences were obtained by using DNAMAN and specific primers were designed and screened by using Primer Premier 5.0.With methods mentioned above, the rapid PCR method for identification of pathogen of Aconiti downy mildew was established. Result: The L1A/L1B primers with high sensitivity and specificity were selected from 8 pairs of designed primers in Primer Premier 5.0.L1A/L1B primers could be used to amplify 670 bp bright bands with detection value from the pathogen DNA, and they can be also used to detect whether the pathogen of Aconiti downy mildew was present in the Aconitum carmichaeli seedling, adult plant and cultivated soil. Conclusion: The primers selected above can be used to detect the pathogen more quickly, easily and effectively, and the established method can be used for the early detection and quarantine of A. carmichaeli seedling with downy mildew.