采用实时荧光定量PCR技术比较提取土壤真菌DNA方法的差异

Comparison of the Difference in the Fungus DNA Extracted with the Artificial Method and Kit Method

[目的]采用实时荧光定量PCR技术比较手提方式和OMEGA微量土壤DNA提取试剂盒获得土壤中真菌DNA的差异。[方法]接种5个梯度稀释的辣椒疫霉菌(Phytophthora capsici)菌丝到灭菌土壤中,并设置不接种的样品为阴性对照。采用手提方式和OMEGA土壤微量DNA提取试剂盒分别提取制备的土壤样品,采用实时荧光定量PCR方法和辣椒疫霉菌的特异性引物对土壤样品中的辣椒疫霉菌进行定量,并对结果进行比较,分析两种方法的特点。[结果]手提方式和试剂盒方式获得的土壤真菌DNA受土壤中杂质的影响较小,并且均能获得适合实时荧光定量PCR技术的模板DNA。采用试剂盒方式获得的辣椒疫霉菌定量结果比手工提取方式获得的辣椒疫霉菌定量结果平均高2.78倍。[结论]采用OMEGA微量土壤DNA提取试剂盒方法获得的菌体基因组DNA的量较多,定量结果更精确;手提方法所需试剂均为常用生化试剂,方便获得且价格低廉。因此,两种方法各有优势,可以根据实际情况选择。

[Objective] The difference in the isolation of fungus DNA with artificial and Kit extracting method was compared based on the test of the real-time quantitative fluoresces PCR technique.[Method] The hyphae of Phytophthora capsici with five different concentrations and the CK was inoculated in the sterile soil medium and the DNA from soil samples was extracted with artificial and OMEGA DNA kit respectively and then the Phytophthora capsici was quantitatively detected with real-time quantitative PCR method and its specific primers.The character of the two isolation DNA methods was analyzed based on the comparison of the result.[Result] It was found that the soil fungus DNA was little affected by other materials in the two methods and the template DNA of real-time PCR technique could be formed.The quantitative results of Phytophthora capsici obtained by Kit method was 2.78 times higher than that obtained in average.[Conclusion] The kit method extracted more DNA and its quantitative results were more accurate;all reagents needed by artificial method were common biochemical reagents,which were affordable and easy to acquire.Therefore,both methods had their own advantages,the choice of them should be based on practical conditions.