兔斯氏艾美耳球虫环介导等温扩增技术检测方法的建立  

Development of Loop-mediated Amplification for Detection of Eimeria stiedai

作　　者：温福利[1] WEN Fu-li(Department of Comparative Medicine, Fuzhou General Hospital of Nanjing Command, PLA, Fuzhou 350025, Chin)

机构地区：[1]福州总医院比较医学科,福州350025

出　　处：《实验动物与比较医学》2018年第3期176-181,共6页Laboratory Animal and Comparative Medicine

基　　金：福建省科技计划引导性项目(2015Y0075);南京军区福州总医院临床应用研究专项(2015L01)

摘　　要：建立检测兔斯氏艾美耳球虫(E.stiedai)环介导等温扩增技术(LAMP)的方法。方法根据E.stiedai的ITS1-5.8s RNA-ITS2基因序列设计特异引物,通过优化反应条件,建立E.stiedai的LAMP检测方法。结果 25μL的反应体系中Bst聚合酶的最优添加量是1.5μL,DNA的最优添加量是75 ng,环引物、内引物和外引物的最优浓度依次是0.8μmol/L、1.6μmol/L和0.15μmol/L,在63℃反应1 h后能特异性地扩增目的基因,并且具有较高的灵敏度。结论建立了E.stiedai的LAMP检测方法,为E.stiedai感染的快速检测提供了新思路。Objective Loop-mediated amplification（LAMP） approaches was developed for detecting Eimeria stiedai（E.stiedai）. Methods Specific primers were designed and synthesized according to a part of the sequence of ITS1-5.8 s RNA-ITS2 region of E.stiedai. The LAMP methods of detecting E.stiedai was established by optimizing the reaction conditions. Results In 25 μL reaction system, the optimal dosage of Bst polymerase is 1.5 μL, and the optimal dosage of DNA is 75 ng. The optimal concentration of ring primers, internal primers and external primers is 0.8 μmol/L, 1.6 μmol/L and 0.15 μmol/L respectively. The target E. stiedai strip can be specifically amplified when reacted for 1 h at 63 ℃. The sensitivity of LAMP was high for determing E.stiedai, and this method could specifically detect the DNA of E.stiedai. Conclusions The LAMP method for detecting E.stiedai has been established,which provides a new method for rapid detection of E. stiedai infection.

关 键 词：兔斯氏艾美耳球虫 PCR 环介导等温扩增技术(LAMP) 敏感性 特异性 

分 类 号：R-332[医药卫生] Q95-33[生物学—动物学]