Chin J Schisto Control ›› 2011, Vol. 23 ›› Issue (5): 558-560.

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

Xu Xiang-zhen, Jin Xiao-lin, Li Jian, Jiang Wen-cai, Jiang Gang   

  1. Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China
  • Online:2011-10-24 Published:2011-10-17

环介导等温扩增技术检测细粒棘球绦虫DNA的初步研究

徐祥珍|金小林|李健|江文才|蒋岗   

  1. 江苏省寄生虫病防治研究所 (无锡 214064)
  • 作者简介:徐祥珍|女|副主任医师。研究方向:蠕虫病控制
  • 基金资助:

    江苏省卫生厅项目(X200929)

Abstract:

Objective To evaluate the sensitivity of loop?mediated isothermal amplification (LAMP) on the detection of Echi? nococcus granulosus. Methods The DNAs were extracted from Echinococcus granulosus eggs and adults. According to Echinococ? cus 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 prod? ucts 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. Electrophore? sis 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.

Key words: Echinococcus granulosus, Loop?mediated isothermal amplification (LAMP), 12S rRNA, Detection

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 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12S rRNA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SYBR Green Ⅰ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1 000、 100、 10、 1个虫卵/200 μl 细粒棘球绦虫虫卵DNA进行LAMP反应, 评价其敏感性。结果 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 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。

关键词: 细粒棘球绦虫, 环介导等温扩增技术, 12S rRNA, 检测

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