土壤真菌分子生态学研究需要大量和高效地提取真菌的 DNA。应用市售的一般试剂盒提取土壤虫生真菌DNA，常常存在得率和质量低的问题，甚至根本提取不到土壤真菌的DNA样品。针对这一问题，本研究对细胞破碎方式及后续DNA提取参数进行了一系列的改进和优化，建立了一套针对性的提取方法。该提取法对虫生真菌—蜡蚧轮枝菌纯培养物的灵敏度极高，可在10个孢子的条件下提取到DNA，而市售的试剂盒则不能提取到（试剂盒在107个孢子条件仍提不到DNA）；该提取法对人工投菌土样的DNA提取灵敏度达到102个孢子/克土，所得的DNA纯度高，无明显抑制后续PCR扩增的物质；施用过蜡蚧轮枝菌的田间土样所得 DNA 样品可成功扩增出蜡蚧轮枝菌特异片段，而未施用过蜡蚧轮枝菌的田间土样所得DNA 样品不能扩增出蜡蚧轮枝菌特异片段。本提取方法具有灵敏度和纯度高的特点，适用于土壤虫生真菌的分子生态学样品制备。%It is required to efficiently extract large quantity and high quality of fungal DNA for the soil fungal molecular ecological study. However, the yield and quality of DNA are often insufficient or even no any product can be produced by the commercial DNA extraction kit. To solve these problems, the cell lysis and subsequent DNA extraction parameters were optimized in this study, and then an efficient method of extracting soil fungal DNA was established. The results showed this method was very sensitive to entomopathogenic fungi Verticillium lecanii. DNA could be extracted from the pure culture samples which contained more than 10 spores, and the artificial soil samples which contained more than 100 spores per gram soil. However commercial DNA extraction kits are unable to extract DNA from the pure culture samples which contained 107 spores. The DNA product from our method did not show any obvious inhibition in the subsequent PCR amplification, and was proved to be high-pure by the OD260/280 value. DNA could be also extracted from - two natural soil samples,but the target sequence fragment specific for V. lecanii was only amplified from one natural soil sample applied with V. lecanii. In conclusion, this soil DNA extraction method is sensitive to entomopathogenic fungi and DNA products were high-pure, so it was suitable for the DNA preparation in soil entomopathogenic fungal molecular ecological research.
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