一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

DNA extraction from yeasts,Prototheca and filamentous fungi for PCR

目的介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法所用菌种包括临床分离的未知菌株和保藏菌株共23株:未知酵母菌(5株)、真皮毛孢子菌(1株)、糠秕马拉色菌(1株)、季也蒙念珠菌(5株)、未知丝状真菌(6株)、无绿藻(1株)、烟曲霉(2株)、拟青霉菌(1株)、茎点霉(1株)。用溶细胞酶(lyticase)结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260/A280检测纯度并计算质量浓度,用真菌通用引物ITS1/ITS4扩增真菌核糖体基因(rDNA)内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。

Objective To introduce a DNA extraction method from yeasts,Prototheca and filamentous fungi for PCR. Methods Clinical isolated and stored strains including unknown yeasts （5 strains） and filamentous fungi （6 strains）, Trichosporon dermatis （1 strain）, Malassezia furfur （ 1 strain ）, Paecilomyces （ 1 strain ）, Phoma （ 1 strain ）, Prototheca （ 1 strain ） , Candida guilliermondii （5 strains） and AspergiUus fumigatus （2 strains） were tested. Lyticase combined with Biospin Fungus Genomic DNA Extraction Kit were used to extract the DNA according to the manufacturer＇s protocol. The DNA purity was checked by analyzes the ratio of A260/A280 and its concentration was calculated. The universal fungal primers ITS1/ITS4 were used for PCR reaction, to amplify ITS region of ribosome RNA gene （rDNA）, by means of the gained DNA as the templates. Results All of 23 tested fungi＇s genomic DNA could be extracted successfully, and the ratio of A260/A280 ranged from 1.6 to 2.0. All of the PCR products appeared clear DNA bands after electrophoresis. Conclusions Combination of lyticase with Biospin Fungus Genomic DNA Extraction Kit is a convenient and feasible way to extract genomic DNA from yeasts,Prototheca and filamentous fungi for PCR reaction.