环介导等温扩增技术检测细粒棘球绦虫DNA的初步研究  

Preliminary study on detection of Echinococcus granulosus DNA by loop-mediated isothermal amplification

作　　者：徐祥珍[1] 金小林[1] 李健[1] 江文才[1] 蒋岗[1] 

机构地区：[1]江苏省寄生虫病防治研究所,无锡214064

出　　处：《中国血吸虫病防治杂志》2011年第5期558-560,565,共4页Chinese Journal of Schistosomiasis Control

基　　金：江苏省卫生厅项目(X200929)

摘　　要：目的评价环介导等温扩增技术(LAMP)检测细粒棘球绦虫的敏感性。方法提取细粒棘球绦虫虫卵及成虫DNA,根据棘球绦虫线粒体12SrRNA基因序列及LAMP法原理,设计4条细粒棘球绦虫特异性引物,进行LAMP反应,反应产物经SYBRGreenⅠ显色及1.5%琼脂糖电泳鉴定,同时设置泡状带绦虫及空白对照。用1000、100、10、1个虫卵/200μl细粒棘球绦虫虫卵DNA进行LAMP反应,评价其敏感性。结果细粒棘球绦虫检测管反应液呈混浊沉淀,显色后为绿色;对照组澄清,显色后为棕色。虫卵产物电泳后呈LAMP特征性梯状条带,对照组无扩增产物。LAMP可检测到的虫卵最低数量为1个。结论 LAMP法敏感、特异、简便,可用于棘球蚴病病原检测和疾病监测。Objective To evaluate the sensitivity of loop-mediated isothermal amplification（LAMP） on the detection of Echinococcus granulosus.Methods The DNAs were extracted from Echinococcus granulosus eggs and adults.According to Echinococcus mitochondrial 12S rRNA sequences and the mechanism of LAMP,4 Echinococcus specific primers were designed and used for LAMP assay,and Bubble taenia and the blank were used as the negative control for evaluation of the specificity.The LAMP products were stained by SYBR Green Ⅰand analyzed by electrophoresis,and 1000,100,10,1 eggs of Echinococcus granulosus per 200 μl were amplified by LAMP for evaluating the sensitivity.Results The LAMP products of Echinococcus granulosus adult DNA became turbid and green after staining while the products of control DNA kept clarify and brown after staining.Electrophoresis analysis showed that the LAMP products of Echinococcus granulosus eggs presented characteristic ladders,but the products of control did not.The detection limit of LAMP assay was 1 egg of Echinococcus granulosus per reaction.Conclusions LAMP assay is a simple,sensitive and specific method for detection of hydatid disease pathogens and could be used for the disease surveillance.

关 键 词：细粒棘球绦虫 环介导等温扩增技术 12S RRNA 检测 

分 类 号：R383.33[医药卫生—医学寄生虫学]