弓形虫环介导等温扩增快速检测方法的建立

Development of the loop-mediated isothermal amplification assay for Toxoplasma gondii

为了建立弓形虫的快速检测方法,本研究建立了一种灵敏、特异、快速的分子生物学检测方法——环介导等温扩增技术(LAMP)。用10倍系列稀释的DNA样品对该检测方法的灵敏度进行了测试,同时通过对扩增产物做内切酶验证;为证明LAMP技术的特异性,对新孢子虫、附红细胞体、瑟氏泰勒虫等病原体进行检测。结果显示:利用LAMP技术成功检测到弓形虫基因区,此方法的灵敏度可达到能检测5个拷贝DNA分子水平,酶切分析结果正确,并且特异性强,对新孢子虫、附红细胞体、瑟氏泰勒虫等病原体检测均为阴性。说明LAMP技术可应用于弓形虫的快速检测。

In order to develop a rapid and simple method for Toxoplasma gondii infection, a novel loop-mediated isothermal amplification （LAMP） assay for T. gondii was established. Ten serial dilutions of DNA sample was used to evaluate the sensitivity of assay, and the accu- racy was demonstrated by incision enzyme. The pathogens, Neospora, Eperythrozoon and Sergenti, were detected in order to evaluate the specificity, The result showed that T. gondii gene could be detected by LAMP, and the detection limit was 5 copies. The result of incision enzyme was correct. The specificity of LAMP assay was good and no amplification product was found in the samples of Neospora, Eperythrozoon and Sergenti. It suggested LAMP assay was feasible for detection of T. gondii.