粮食真菌DNA快速高通量提取方法的建立与应用

Establishment and application of a rapid high-throughput extraction method of fungal DNA for grains

中文关键词:  真菌  DNA提取  磁珠  粮食  脱氧雪腐镰刀菌烯醇  镰刀菌

英文关键词:fungal  DNA extraction  magnetic beads  grain  deoxynivalenol  Fusarium

中文摘要:

      目的 建立一种高效、快速、高通量的粮食中真菌DNA提取和分析方法, 并应用于小麦产脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)镰刀菌污染状况研究。方法 基于磁珠纯化技术, 开发了一种粮食基质中真菌DNA的快速提取方法—月桂酰肌氨酸钠(sodium lauroyl sarcosine, SLS)磁珠法, 结合前期采用实时荧光定量聚合酶链式反应法(quantitative real-time polymerase chain reaction, qPCR)和微滴式数字PCR法(droplet digital PCR, ddPCR)建立的小麦中3-乙酰基脱氧雪腐镰刀菌烯醇(3-acetyldeoxynivalenol, 3ADON)和15-乙酰基脱氧雪腐镰刀菌烯醇(15-acetyldeoxynivalenol, 15ADON)两种产DON毒素化学型镰刀菌及禾谷镰刀菌复合群分析方法, 对其提取效果进行了验证并分析了我国2021年新收获小麦产DON镰刀菌的污染水平。结果 与十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)法、柱式法试剂盒和磁珠法试剂盒相比较, 本研究建立的SLS磁珠法裂解过程无需水浴, 配合自动化提取设备工作, 整个提取时间小于1 h。对禾谷镰刀菌复合群的提取率最优, 而对3ADON和15ADON化学型镰刀菌的提取率与经典CTAB法、磁珠法试剂盒没有显著性差异, 并且显著优于柱式法试剂盒。在对2021年收获的120个小麦样品监测结果显示, 产DON镰刀菌的生物量与DON毒素水平之间具有极显著的相关性(P<0.01)。结论 SLS磁珠法适用于从基质复杂的粮食样品中提取真菌DNA, 操作简便快速、提取率高, 更易实现大样本量的分子生物学准确定量检测需求。

英文摘要:

      Objective To establish an efficient, rapid and high-throughput method for the extraction and analysis of fungal DNA in grains and apply to study the contamination status of deoxynivalenol (DON) producing Fusarium in wheat. Methods Based on the magnetic bead purification technology, a rapid extraction method of fungal DNA from grain substrate—sodium lauroyl sarcosine (SLS) magnetic bead method was developed. Combined with the 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) in wheat established by quantitative real-time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) 2 kinds of methods for analysis of chemical Fusarium producing DON toxin and Fusarium graminearum complex, the extraction effect was verified and the contamination level of DON producing Fusarium in newly harvested wheat in 2021 in China was analyzed. Results Compared with the cetyltrimethylammonium bromide (CTAB) method, column method kit and magnetic bead method kit, the SLS magnetic bead method established in this study needed no water bath heating in the cracking process in about 1 h with the automated extraction equipment. The SLS magnetic bead method performed best in the extraction rate of the Fusarium graminearum species complex among the DNA extraction methods mentioned above, and the extraction rate of 3ADON and 15ADON chemical types of Fusarium had no significant difference from the CTAB method and magnetic bead method kit, and was significantly better than the column method kit. The monitoring results of 120 wheat samples harvested in 2021 showed that there was a very significant correlation between the biomass of DON producing Fusarium and the level of DON toxin (P<0.01). Conclusion The SLS magnetic bead method is suitable for extracting fungal DNA from grain samples with complex matrix, with simple operation and high extraction rate, which makes it easier to meet the requirements for accurate quantitative detection of molecular biology with large sample size.