逆转录环介导等温扩增技术检测H3亚型猪流感病毒  

Rapid Detection of H3 Subtype Swine influenza virus by RT-Loop-Mediated Isothermal Amplification

作　　者：刘中勇[1] 朱道中[1] 李少璃[2] 冀君[2] 毕英佐[2] 谢青梅[2] 曹永长[2] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广东广州510623 [2]华南农业大学动物科学学院,广东广州510642

出　　处：《动物医学进展》2009年第7期1-5,共5页Progress In Veterinary Medicine

基　　金：广东省重大科技专项(2005A20901004;2006A20901003)

摘　　要：基于环介导等温扩增技术(RT-LAMP),建立了一种用于快速检测H3亚型猪流感病毒的方法。根据GenBank中收录的H3N2亚型猪流感病毒(SIV)HA基因保守区的序列,设计一组对应HA序列6个区域的4条特异性引物,建立RT-LAMP反应体系;经优化,确定了最佳的LAMP反应条件。扩增后产物形成的焦磷酸离子和溶液中的镁离子结合而形成焦磷酸镁沉淀,结果可视。同时用特异性内切酶HhaⅠ对所获得的产物进行酶切鉴定,所得的酶切产物大小与理论值完全符合。用已建立的RT-LAMP技术检测临床样品,检测结果与RT-PCR方法符合率均达到100%。结果表明,本研究建立的RT-LAMP检测猪流感病毒方法,操作简单,灵敏度高,特异性强,是一种适用于现场诊断的检测技术。In this study, an RT-LAMP （Reverse Transcription Loop-mediated Isothermal Amplification） assay was developed to rapidly detect H3 subtype SIV,and compared with RT-PCR method in accuracy, sensitivity and specificity. A set of four specific primers were designed to recognize six distinct genomic sequences according to the conserved region of the H3 subtype SIV HA hemagglutinin gene,including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 65 ℃ and 60 min, respectively. Comparative experiments showed that RT- LAMP assay is superior to RT-PCR assay and virus isolation in sensitivity and celerity. In addition, enzymatic digestion of RT-LAMP products and cross-specificity test indicated that the newly developed RT- LAMP method is a simple, rapid, accurate, sensitive and specific method for detecting H3 subtype SIV. RT-LAMP assay would be a good alternative method for on-farm disease diagnosis.

关 键 词：H3亚型猪流感病毒 血凝素 逆转录环介导等温扩增 

分 类 号：S852.659.5[农业科学—基础兽医学]