环介导等温扩增快速检测临床假丝酵母菌属方法建立与应用  

Establishment and application of loop-mediated isothermal amplification for rapid detection of clinical isolates of Candida spp

作　　者：鲁勇 汪一萍 应建飞 俞燕红 贺明阳 

机构地区：[1]宁波市鄞州人民医院检验科,浙江宁波315040

出　　处：《中华医院感染学杂志》2015年第6期1204-1206,共3页Chinese Journal of Nosocomiology

基　　金：宁波市自然科学基金资助项目(2011A610047)

摘　　要：目的：建立一种环介导等温扩增（LAMP）反应，根据假丝酵母菌属 D1～ D226S rDNA 保守区域设计通用引物，直接对临床标本进行假丝酵母菌属检测。方法根据假丝酵母属的特异 D1～ D226S rDNA 基因序列比对后的相对保守区设计特异引物，分别提取5种假丝酵母菌、临床对照菌株以及临床标本 DNA ，然后以 LAMP 技术扩增，并对白色假丝酵母菌孢子悬液进行系列梯度稀释检测，确定最低检测限。结果5种假丝酵母菌直接提取 DNA 经 LAMP 和 PCR 方法检测均为阳性；30份经培养证实假丝酵母菌属感染标本，经 LAMP 检测有27份标本阳性，阳性检出率为90．0％；另10份临床对照标本和11种非假丝酵母菌属来源 DNA 样本经 LAMP 方法检测后结果均为阴性，无非特异性反应发生；LAMP 最低检测限达到103 CFU ／ml 。结论结果证明 LAMP 是一种简便、快速、特异和敏感的检测方法，可以检测临床和环境标本中的常见假丝酵母菌属。OBJECTIVE To establish the loop-mediated isothermal amplification for direct detection of Candida spp in clinical samples by designing universal primers on the basis of conserved region of D - D2 26S rDNA of Candida spp .METHODS The specific primers were designed from the relative conservation region of the published sequence of D1-D2 26S rDNA genes of the Candida sp p ,then the genomic DNA was extracted from five species of Candida spp ,clinical control strains ,and clinical specimens and was amplified by using loop-mediated isothermal amplifica-tion ,and the serial dilutions were carried out for the spore suspensions of Candida albicans so as to determine the minimum detection limit .RESULTS The loop-mediated isothermal amplification and PCR assays obtained positive results for the DNA that was directly extracted from the five species of Candida sp p .Of 30 specimens that were proved infected with Candida sp p through culture ,27 were tested positive by the loop-mediated isothermal ampli-fication ,with the positive detection rate of 90 .0% .While 10 clinical control specimens and 11 DNA samples of non-Candida were tested negative by the loop-mediated isothermal amplification ,and the non-specific reactions did not occur .The minimum detection limit of the loop-mediated isothermal amplification reached to 103 CFU /ml . CONCLUSION It is concluded that the loop-mediated isothermal amplification is a simple ,rapid ,specific ,and sen-sitive method for detection of common Candida sp p in clinical and environmental specimens .

关 键 词：假丝酵母菌属 环介导等温扩增 核酸 

分 类 号：R379.4[医药卫生—病原生物学]