一种简便、快速、高效适合PCR的真菌DNA提取方法

摘要

目的 探索一种新的真菌DNA提取方法,该方法操作简便,提取效率高,耗时较短,应用范围广.方法 将酶消化和Chelex-100提取DNA方法联合起来,配制一种新的DNA提取试剂.通过真菌ITS4和ITS5通用引物进行PCR,对新的DNA提取试剂同市售的Takara lysis buffer在DNA提取方面的效能进行评价,同时对DNA浓度和纯度进行测定.结果 实验所用34株真菌,自配试剂成功提取出32株真菌DNA;Takara lysis buffer成功提出27株真菌DNA.对于两种方法都没能提出DNA的2株真菌,经液氮研磨破壁后,自制试剂均成功提取;而Takara lysis buffer仅成功提取DNA 1株.结论 同市售试剂Takara lysis buffer比较,自配DNA提取液提取DNA质量相对较高,可用于临床常见感染真菌的PCR诊断.对于某些真菌,提取DNA之前先进行菌体破壁是必要的.%Objective To explore a new convenient and efficient fungi DNA extraction method,which could be used in many kinds of fungi.Methods We associated enzymic digestion with Chelex-100 to make up a new reagent for fungi DNA extraction.Results During the experiment,34 strains were tested.The DNAs of 32 strains could be obtained by our reagent,while only 27 strains' DNAs could be extracted by Takara lysis buffer.For the strains we did not successfuly obtain the DNA by the above methods,we grinded the fungi by liquid nitrogen.After that,our reagent obtained their DNA by,and only one by Takara lysis buffer.Conclusion The DNA extraction efficiency of our reagent is much better than that of Takara lysis buffer.It can be used in many kinds of fungi.For some fungi,we should firstly grind it before DNA extraction.