羊口疮病毒环介导等温扩增快速检测方法的建立及应用  

Establishment and application of a loop-mediated isothermal amplification method for rapid detection of orf virus

作　　者：向智龙[1] 卓建华 鲜思美[1,3] 欧德渊[1,3] 李启发[1] 徐雄真[1] 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]京山县畜牧兽医局,湖北京山431800 [3]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国兽医科学》2011年第6期588-592,共5页Chinese Veterinary Science

基　　金：贵州省科学技术基金项目(黔科合J字[2010]2260);贵州大学引进人才科研项目(贵大人基合字[2009]024号);贵州大学2010年研究生创新基金项目(校研农[2011]024)

摘　　要：参照GenBank中登录的羊口疮病毒(ORFV)序列(Gu320351),针对OrfV的B2L基因保守区域设计了4条引物,通过优化反应条件,建立了检测OrfV的环介导等温扩增技术(LAMP),并进行了特异性试验、敏感性试验及临床样本检测。结果显示,用建立的LAMP方法对OrfV阳性样品扩增产物的电泳呈特征性梯状条带,而绵羊痘病毒、蓝舌病病毒、口蹄疫病毒、丝状支原体山羊亚种、山羊痘病毒及健康山羊皮肤的扩增产物电泳后均无扩增条带。建立的LAMP方法对OrfV的最低检出量为5.3fg,比常规PCR方法高1 000倍。表明建立的LAMP方法特异性强,敏感性高。对6份临床样本的检测结果显示,OrfV阳性检出率为83.33%(5/6),与常规PCR的符合率为100%。本试验为羊口疮病例的临床诊断和OrfV的快速检测提供了新的方法。The objective of this study was to establish a loop-mediated isothermal amplification（LAMP） method for the detection of orf virus（OrfV）.According to OrfV sequence available in GenBank,a set of four primers were designed based on the OrfV B2L gene sequence.Optimal conditions,specificity test and sensitivity test were performed.In result,ladder-like products were amplified from the OrfV-positive samples by LAMP,while no product was generated from goatpox virus,sheeppox virus,bluetongue virus,foot-and-mouth disease virus,and contagisas caprine pleuropneumonia.The LAMP assay had a detection limit equivalent to 5.3 fg of OrfV,which was 1 000 times more sensitive than PCR method.Tests of 6 clinical samples by the assay showed OrfV-positive rate of 83.33%（5/6）.The agreement rate between LAMP and PCR was 100% in detection clinical samples.In conclusion,the developed LAMP is a novel method for the rapid detection of OrfV.

关 键 词：羊口疮病毒 环介导等温扩增技术 检测 

分 类 号：S852.659.1[农业科学—基础兽医学]