口蹄疫病毒实时TaqMan荧光定量RT-PCR检测方法的建立  

Development of a Real-Time TaqMan RT-PCR Assay for the Detection of Foot-and-Mouth Disease Virus

作　　者：卢受昇[1,2] 樊惠英[1] 孙彦伟 任涛[1] 卢洪芬 闫晓菊 孔令辰 查云峰 廖明[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东省动物防疫监督总所,广东广州510230

出　　处：《华南农业大学学报》2009年第3期86-89,共4页Journal of South China Agricultural University

基　　金：国家自然科学青年基金(30800826);广东省博士启动基金(8451064201001131)

摘　　要：基于口蹄疫病毒聚合酶3D蛋白基因的序列分析,设计合成了特异的引物和探针,通过反应条件的优化,建立了实时TaqMan荧光定量RT-PCR检测方法.试验表明,实时TaqMan荧光定量RT-PCR能特异性检测A、O、Asia I 3种血清型的口蹄疫病毒,对猪水泡病、猪瘟、蓝耳病等猪常见病原检测结果均为阴性.对比检测试验表明,实时Taq-Man荧光定量RT-PCR的检测敏感性比常规的多重RT-PCR提高达105倍,对口蹄疫病毒细胞增殖病毒液的检测灵敏度可达0.063个TCID50.对临床样品的检测试验证实,该方法可以有效检测临床样品中的猪水泡皮、组织、血清及O-P液中的口蹄疫病毒.In this study, one set of primer and one probe were designed according to the sequence of 3D gene of foot-and-mouth disease virus （FMDV）. Through the optimization of reaction conditions, a Taq-Man RT-PCR detection method was developed. The TaqMan RT-PCR assay specifically detected A, O, Asia I serotype of FMDV and the test results were negative for swine vesicular disease, classical swine fever virus,porcine reproductive and respiratory syndrome. Comparison tests indicated that TaqMan RTPCR assay was at least 105 fold sensitive than the conventional multi-RT-PCR. The detection limit of the assay reached 0. 063 TCID50 as showed by tests on serial ten fold dilution of cell propagated virus samples. Clinical applications showed that this method can be used for FMDV detection of swine blisters, tissues, serum, oesophageal-pharyngeal fluid. So it provided one reliable method for the diagnosis and supervision of foot and mouth disease.

关 键 词：口蹄疫病毒 荧光定量RT—PCR 病毒检测 TAQMAN探针 

分 类 号：S852.65[农业科学—基础兽医学]