乙型脑炎病毒可视化快速检测方法的建立  

Establishment of a rapid and visual detection method for Japanese encephalitis virus

作　　者：邢国珍[1] 姬凯 薛继初 徐朝[1] 郑文明[1] 许君[1] XING Guozhen;JI Kai;XUE Jichu;XU Zhao;ZHENG Wenming;XU Jun(College of Life Science,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学生命科学学院

出　　处：《河南农业大学学报》2019年第6期912-917,共6页Journal of Henan Agricultural University

基　　金：国家自然科学基金-河南联合基金项目(U1804108);河南省高等学校重点科研项目计划(19A230005);河南省科技攻关重点项目(132102310033);河南省科技攻关重点项目(162102310439; 172102310437)

摘　　要：利用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)建立了简便、可靠的快速检测乙型脑炎病毒JEV(Japanese encephalitis virus)的可视化方法。根据GenBank数据库中JEV Henan-09-03毒株(GenBank ID:GQ336809.1)E蛋白序列,设计了3对特异性LAMP引物,建立Bst DNA Polymerase等温扩增体系,优化后的反应体系可高灵敏性、特异性检测JEV。本方法通过在扩增产物中加入SYBR Green I染料后用石蜡进行封闭,在可见光下根据反应液荧光的颜色深浅来判断JEV扩增阳性强弱。结果显示,该方法的灵敏度可达5 copies·mL-1,且与其他病毒,如乙型肝炎病毒HBV(Hepatitis B virus,HBV)无交叉反应。A rapid,simple,reliable and visual method for detecting Japanese encephalitis virus(JEV)was established by loop-mediated isothermal amplification(LAMP).Based on the E protein sequence of JEV Henan-09-03 strain(GenBank ID:GQ336809.1)in GenBank database,three specific pairs of LAMP primers were designed and the isothermal amplification system of Bst DNA Polymerase was established.The optimized reaction system could detect JEV virus with specificity and sensitivity.The SYBR Green I dye was added to the amplification product obtained in the reaction system and blocked with paraffin.Under the visible light,the JEV virus could be detected according to the color of the reaction liquid.The results show that the sensitivity of this LAMP method can reach 5copies·mL-1 for JEV and has no cross-reaction with other viruses such as HBV(Hepatitis B virus).

关 键 词：乙型脑炎病毒 E蛋白 环介导等温扩增技术(LAMP) RT-LAMP 特异性 灵敏度 

分 类 号：R373.31[医药卫生—病原生物学]