适用于提取新生隐球酵母菌及白色假丝酵母菌总核糖核酸快速方法研究  

A study on total RNA isolation by fast Abstraction from Cryptococcus neoformansand Candida albicans

作　　者：徐赤宇[1] 刘翠杰[1] 吴建华[2] 仇芸[3] 赵瑾[3] 朱红梅[3] 温海[3] 

机构地区：[1]济南军区总院皮肤科,山东济南250031 [2]第二军医大学附属长海医院皮肤科,上海200433 [3]第二军医大学附属长征医院皮肤科,上海200003

出　　处：《中华医院感染学杂志》2012年第24期5460-5462,共3页Chinese Journal of Nosocomiology

基　　金：第四十七批中国博士后科学基金面上二等资助(20100471802)

摘　　要：目的探讨快速可以兼顾提取高质量的新生隐球酵母菌和白色假丝酵母菌总核糖核酸(RNA)的试验方法,为各种功能基因的表达研究奠定基础。方法针对新生隐球酵母菌荚膜株和荚膜缺陷株新生隐球酵母菌H99及白色假丝酵母菌国际标准菌株ATCC 64548采用冷酸洗玻璃珠联合Yeast RNA kit法提取总核糖核酸(RNA),用紫外线分光光度计测量其OD260、OD280的值,并且进行琼脂糖凝胶电泳,同时应用RT-PCR鉴定RNA质量。结果提取的新生隐球酵母菌荚膜株和荚膜缺陷株总RNA产量分别为0.6μg/107细胞、1.5μg/107细胞,且OD260/OD280比值在1.8～2.0,电泳显示提取的总RNA呈现28SrRNA,18SrRNA和5srRNA 3条清晰的条带;提取的白色假丝酵母和菌丝相总RNA产量分别为2.3μg/107细胞、3.1μg/107细胞,且OD260/OD280比值在1.8～2.0,电泳显示提取的总RNA呈现28SrRNA、18SrRNA和5SrRNA 3条清晰的条带。结论冷酸洗玻璃珠联合Yeast RNA kit法,快速高质量适用于大小样本的新生隐球酵母菌和白色假丝酵母菌总RNA提取,满足各种类型分子生物学研究需要以及临床鉴别诊断治疗不同种类真菌感染。OBJECTIVE To investigate the total RNA isolation of Cryptococcus neoformans and Candida albicans by fast Abstraction so as to lay the foundation for the study on the expression of a variety of functional genes.METHODS The total RNA of Cryptococcus neoformans（one with capsule and one with deficient capsule（H99）） and C.albicans ATCC64548 were isolated by Yeast RNA kit combined with modified glass beads method.The values of OD260 and OD280 were measured by ultraviolet spectrophotometer and agarose gel electrophoresis,and the quality of RNA was determined with RT-PCR.RESULTS The outputs of total RNA from Cryptococcus neoformans were 0.6 μg/107 and 1.5 μg/107 cells,respectively,the ratio of OD260/OD280 was between 1.8 and 2.0.The result of electrophoresis showed that there appeared three clear bands 28S rRNA,18S rRNA and 5S rRNA.The outputs of total RNA from C.albicans were 2.3 μg/107 and 3.1 μg/107 cells,respectively,the ratio of OD260/OD280 was between 1.8 and 2.0.The result of electrophoresis showed that there appeared three clear bands 28S rRNA,18S rRNA and 5S rRNA.CONCLUSION The modified glass beads combined with Yeast RNA kit method is suitable for extraction of RNA from a variety of Cryptococcus neoformans and C.albicans,having met the needs of the various types of molecular biology researches and the clinical identification,diagnosis and treatment of various fungal infections.

关 键 词：新生隐球酵母菌 白色假丝酵母菌 总核糖核酸 YeastRNAkit法 玻璃珠法 

分 类 号：R379[医药卫生—病原生物学]