猪流行性乙型脑炎RT-LAMP方法的建立与初步应用  

The Construction and Application of the Method of RT-LAMP for Swine Japanese Encephalitis

作　　者：卢冰霞[1,2] 刘磊[2] 秦毅斌[1] 赵武[1] 梁家幸[1] 郭旋[2] 黎木兰[2] 李斌[1] 何颖[1] 苏乾莲[1] 梁保忠[1] 闭炳芬[1] 黄伟坚[2] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西大学动物科学技术学院,广西南宁530005

出　　处：《安徽农业科学》2013年第12期5359-5361,5365,共4页Journal of Anhui Agricultural Sciences

基　　金：广西科技攻关项目(桂科攻1123007-3);公益性行业(农业)科研专项(201103008;2008030152GX);广西基本科研业务费专项(桂科专项13-1);广西自然科学基金项目(2011GXNSFB018032);广西畜禽疫苗新技术重点实验室系统性研究课题(12-071-28-A-5)

摘　　要：[目的]建立1种快速、灵敏、特异的检测猪流行性乙型脑炎病毒(JEV)逆转录环介导等温扩增(RT-LAMP)检测方法。[方法]根据JEV E基因序列的保守区6个位点设计了4条特异性LAMP引物,以JEV阳性样品RNA为模板进行一步法扩增,并对反应条件和反应体系进行了优化。[结果]该方法具有较高的灵敏度,其检测极限为0.5 pg;特异性试验表明其具有较高的特异性,对其他病毒性病原体均无扩增反应。与RT-Nested-PCR相比,2种方法检出率的符合率为90.9%。反应结束后可以通过添加化学发光剂进行可视化观察,大大缩短了检测时间。该方法无需特殊仪器,只需在水浴锅中进行,是一种适于基层的简便、灵敏、快速的猪JE的检测方法。[结论]成功建立了猪流行性乙型脑炎RT-LAMP方法,并在临床上进行了初步应用。[Objective] To establish a rapid,sensitive and specific detection method which is reverse transcription loop-mediated isothermal amplification（RT-LAMP） detection method for porcine Japanese encephalitis virus（JEV）.[Method] Four specificity LAMP primers were designed according to the conserved region of the six loci of the JEV E gene sequence,JEV positive sample RNA as a template for one-step amplification,and the reaction conditions and the reaction system was optimized.[Result]The results showed that the method had high sensitivity,the detection limit was 0.5 pg;specificity experiments proved that with high specificity and there was no amplification reactions for other viral pathogens.Compared with the RT-Nested-PCR,the detection rate was 90.9%.And by adding a chemiluminescent agent after the end of the reaction,could conduct visual observation,greatly reducing the detection time.This method did not require special equipment,just in the water bath,it will be a sensitive and rapid detection method for swine JE,that should be a simple suitable for grassroots.[Conclusion] A swine Japanese encephalitis RT-LAMP method was successfully established,and preliminarily used in clinical.

关 键 词：猪流行性乙型脑炎病毒 RT-LAMP 敏感性 特异性 

分 类 号：S852.65[农业科学—基础兽医学]