摘要：
目的 比较不同方法提取真菌基因组DNA的优劣,寻找一种用于PCR扩增的快速、高效的真菌DNA提取方法.方法 分别用加热裂解法、微波法、反复冻融法、溶壁酶法、蜗牛酶法和试剂盒法提取浙江中医药大学附属杭州第一医院保存的马尔尼菲青霉、小孢根霉、新型隐球菌和白假丝酵母菌DNA,在凝胶成像系统下观察基因组DNA电泳图.采用超微量核酸蛋白测定仪测定基因组DNA的浓度和纯度,并计算产率;同时对提取的基因组DNA进行PCR扩增并测序.采用方差分析和SNK-q检验比较不同方法所得基因组DNA浓度和产率.结果 6种方法提取马尔尼菲青霉(霉菌相和酵母相)、小孢根霉、新型隐球菌和白假丝酵母菌所得DNA浓度和产率差异均存在统计学意义(F=750.83,220.95,669.35,132.01,510.20和1658.35,287.10,963.64,1147.77,4521.22,P值均＜0.01),且微波法所得真菌DNA浓度和产率最高,加热裂解法次之,试剂盒法最低.所有方法所得DNA经PCR扩增均出现阳性条带.结论 6种方法均可成功提取真菌基因组DNA,微波法和加热裂解法操作简便,所得真菌基因组DNA模板能够满足临床实验室PCR扩增需求.%Objective To identify a rapid and efficient fungal genomic DNA extraction method for PCR amplification.Methods Genomic DNA was extracted from Penicillum marneffei,Rhizopus microsporus,Cryptococcus neoformans and Candida albicans by heating pyrolysis,microwave,repeated freezing and thawing,lysozyme digestion,overnight snail enzymatic and Qiagen kit methods.DNA electrophoretogram was observed by gel imaging system.The concentration and purity of extracted DNA were determined with an ultramicro nucleic acid protein tester and the yields were calculated.PCR amplification and sequencing were also performed.ANOVA and SNK-q test were used for data analysis.Results There were statistical differences in concentrations and yields of the fungal DNA extracted from Penicillum marneffei (hyphal phase and yeast phase),Rhizopus microsporus,Coptococcus neoformans and Candida albicans by six methods (F=750.83,220.95,669.35,132.01,510.20 and 1658.35,287.10,963.64,1147.77,4521.22,all P ＜0.01).Of six methods,microwave method gained the highest DNA concentration and yield,followed by heating pyrolysis method,while Qiagen kit method obtained the lowest concentration and yield.All DNA extracted by 6 kinds of methods were positive in PCR amplification.Conclusion All of the six methods can be used for fungal DNA extraction which is sufficient for PCR amplification,but microwave and heating pyrolysis methods are more easy and simple to perform.
