流行性乙型脑炎病毒一步法RT-LAMP检测方法的建立  

Development of one step RT-loop-mediated isothermal amplification assay for detection of Japanese encephalitis virus

作　　者：杜鹃[1,2] 宋永[1] 刘立科[2] 华荣虹[2] 

机构地区：[1]黑龙江大学生命科学学院,黑龙江哈尔滨150080 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2010年第10期773-776,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30700027);公益性行业科研专项(200803015)

摘　　要：为建立一种简便、快速、灵敏、特异的乙型脑炎病毒(JEV)检测方法,本研究根据GenBank中登录的25株流行性JEV基因组序列,设计3对特异性的环介导等温扩增(LAMP)引物,优化反应体系,建立了一步法反转录-LAMP(RT-LAMP)检测方法。该方法检测时间短,灵敏度比常规RT-PCR方法高,可检测出103TCID50/0.2mL JEV;特异性强,对猪细小病毒、猪圆环病毒、伪狂犬病毒、猪瘟病毒等常见猪病毒均无交叉反应。结果判定时只需要在扩增产物中加入SYBR GreenⅠ染料,就可以直接在可见光或紫外光下观察颜色变化。该方法简便快捷,省时省力,是一种适用于基层实验室快速、准确检测流行性JEV的方法。To develop a rapid and sensitive method for detecting Japanese encephalitis virus（JEV）,an one-step reverse transcription loop-mediated isothermal amplification（RT-LAMP） assay was established using three pairs of specific primers derived from the genome sequences of 25 different JEV strains.The RT-LAMP assay could detect 103 TCID50/0.2 mL of JEV,approximately 10 times more sensitive than RT-PCR.It was highly specific for JEV and had no crossing-reaction with other swine viruses including Porcine parvovirus,Porcine circovirus,Pseudorabies virus and Classical swine fever virus.The assay could be complete within two hours and the results visualized directly or under the UV light with added SYBR Green I dye.Therefore the RT-LAMP assay developed in this study provided a rapid and practical method for JEV detection.

关 键 词：流行性乙型脑炎病毒 反转录环介导等温扩增技术 

分 类 号：S852.65[农业科学—基础兽医学]