草菇培养料中微生物总DNA的高效提取

Efficient Extraction and Purification of Total Microbial DNA from the Substrate for Straw Mushroom

比较了化学裂解与酶裂解相结合法、氯化苄法、CTAB法、改良CTAB法、MP土壤试剂盒法和植物试剂盒法6种提取草菇培养料总DNA的方法。结果表明:不同方法的A260/A280比值有较大差异,其中化学裂解与酶裂解相结合法提取DNA的产量最高,而新型植物基因组DNA提取试剂盒提取培养料DNA的产量最低,分别为(2 372.72±28.85)μg/g和(87.67±2.04)μg/g。采用真菌、细菌及放线菌特异性引物进行PCR扩增,结果表明,除氯化苄法没有扩增出目的条带外,其余方法均能扩增出目的条带,说明其均能从草菇培养料中同时有效地提取真菌、细菌和放线菌总DNA。综合评价了各方法的DNA产量、纯度、PCR扩增效果、试验时间、费用成本5个指标,其中采用化学裂解与酶裂解相结合法提取出的DNA产量最高,但所用时间较长;MP土壤试剂盒提取的DNA纯度最高、所需时间较短,但成本较高。

An experiment was conducted to compare six methods of DNA extraction of straw mushroom（ Volvariella volvacea）, namely combined extraction method of chemical lysis and enzyme lysis, benzyl chloride method, Cetyl trimethylammonium bromide （CTAB） method, modified CTAB method, Fast DNA Spin Kit for Soil and Plant DNA Kit. The concentrations of DNA （A260/A280） extracted by those six methods were different. Among them, the yied of DNA extracted by the combined extraction method of chemical lysis and enzyme lysis was the highest （ （2372.72±28.85）μg/g）, while that extracted by Plant DNA Kit was the lowest （（87.67±2.04）μg/g）. The DNA extracted by the six methods was amplified by PCR using specific primers of fungi, bacteria and actinomycetes. Results showed that the target strip was detected in all those methods except the benzyl chloride method. Five indexes, including DNA yield, DNA purity, PCR amplification efficiency, extraction time and cost were evaluated. The yield of DNA extracted by the combined extraction method of chemical lysis and enzyme lysis was the highest, but the extraction time was longer. The purity of DNA extracted by Fast DNA Spin Kit for Soil was the best and the extraction time was shorter, but the extraction cost was higher.