多重PCR在快速检测常见假丝酵母菌中的应用  

Application of multiplex PCR in the rapid detection of 6 common Candida species

作　　者：吴媛[1] 陈霞[2] 车洁[1] 边富宁[1] 李文革[1] 卢金星[1] 

机构地区：[1]中国疾病预防控制中心传染病所,北京102206 [2]首都医科大学附属复兴医院,北京100038

出　　处：《中国卫生检验杂志》2014年第22期3247-3250,共4页Chinese Journal of Health Laboratory Technology

基　　金：中国疾病预防控制中心青年科研基金(2012A107);国家自然科学基金青年基金(81301409)

摘　　要：目的建立临床常见感染假丝酵母菌多重PCR快速检测法。方法根据假丝酵母菌r DNA序列,设计光滑假丝酵母、白念假丝酵母和近平滑假丝酵母的种特异性引物,并应用真菌通用引物ITS1和ITS4,以白念假丝酵母、光滑假丝酵母、热带假丝酵母、克柔假丝酵母、近平滑和季也蒙假丝酵母共6种菌的DNA为模板,进行多重PCR,2%琼脂糖凝胶电泳检测结果。大肠杆菌,金黄色葡萄球菌和铜绿假单胞菌为阴性对照。同一PCR反应均重复2次。实验室保存的77株假丝酵母菌(25株白念假丝酵母,15株光滑假丝酵母,15株热带假丝酵母,8株克柔假丝酵母,8株近平滑假丝酵母,6株季也蒙假丝酵母)检测其重复性和稳定性。结果得到3条种特异性引物,扩增出种特异性条带和ITS区域片段。重复2次均得出同样大小的产物片段。阴性对照均未扩增出条带。结论该方法能快速准确检测6种常见感染的假丝酵母菌,具有高特异性。与传统培养方法相比,提高了非白假丝酵母鉴定的准确性,缩短了检测时间,具有一定的临床应用价值。Objective To set up the rapid detection of common infected Candida by multiplex PCR.Methods According to the rRNA sequences of Candida,species-specific primers of Candida glabrata,Candida tropicalis and Candida parasilosis were designed.The multiplex PCR was performed using the 6 C.albicans,C.krusei,C.glabrata,C.tropicalis,C.parasilosis and C.guilliermondii.PCR was done using Candida genomic DNA as template,the species-specific primers and universal yeast primer of ITS region.The PCR products were checked by electrophoresis on 2% agarose gel.Results The species-specific primers of C.albicans,C.krusei and C.parasilosis were designed respectively.Specific DNA band and species-specific ITS DNA fragments were produced by multiplex PCR.Every multiplex PCR reaction was repeated twice and got the same PCR fragment of each species.No DNA fragments were produced in negative control.Expected DNA fragments were got when tested with Candida strains conserved in our lab.Conclusion This multiplex PCR method could identify the common Candida stains to 6 species level rapidly with high specificity.Compared with traditional culture method,this method raised the accuracy of identification for non-albicans Candida,and shorted the consuming time.It may be a potential way applied for rapid identification of clinical Candida strains.

关 键 词：多重引物聚合酶链式反应 假丝酵母菌 快速检测 

分 类 号：R379.9[医药卫生—病原生物学]