食源性动物组织中CSFV和PRRSV双重双色荧光定量RT-PCR检测方法的建立及应用  

Development of the duplex real-time RT-PCR based on dual labeled fluorescent probes for detection of classical swine fever virus and porcine reproductive and respiratory syndrome virus from food-oriented pig tissues

作　　者：魏文康[1] 吕殿红[1] 温肖会[1] 罗胜军[1] 黄忠[1] 周秀蓉[1] 贾春玲[1] 袁洁[1] 

机构地区：[1]广东省农业科学院兽医研究所广东省兽医公共卫生实验室,广东广州510640

出　　处：《中国预防兽医学报》2011年第8期620-624,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省科技计划项目农业攻关(2009B020307007);广州市科技计划项目重大科技专项(2009A1-E041-1);广东省科技计划项目促进服务业发展专项计划(2010A040207006)

摘　　要：为建立同时检检测食源性动物组织中猪瘟病毒(CSFV)和猪蓝耳病病毒(PRRSV)的双色荧光定量RT-PCR方法。本研究根据SCFV和PRRSV基因序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应的体系和扩增条件,建立了能够检测食源性动物组织中SCFV和PRRSV的双重双色荧光定量PCR的方法。其检测下限为1×102拷贝/μL,而且与其他一些猪病病毒无交叉反应,具有良好的特异性。该方法重复性和稳定性试验表明其组内和组间的变异系数最高分别为4.2和4.5。对比试验表明,该方法对CSFV和PRRSV检验的敏感性为常规RT-PCR方法的200倍;该方法的建立为食源性动物组织中CSFV和PRRSV提供了有效手段,该方法特异性和敏感性较好,能够应用于临床检测。To establish the method of real-time PCR for detection of classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） in food-oriented animal tissues, the specific primers and TaqMan fluorescent probes of CSFV and PRRSV were designed and synthesized, and the amplification systems were optimized. The duplex real-time RT-PCR was established based on dual labeled FAM or VIC fluorescent probes. The method established in this study showed better amplification efficiency in detecting CSFV and PRRSV with detection limit of 10^2 copies, which was 200 times more sensitive than the normal PCR methods and no any reaction with other related swine viruses. The intro-and inter reproducibility were 4.2% and 4.5%, respectively. Tested on 200 clinical samples by this method showed a complete coincidence with other detection methods. This specificity and sensitivity method could be used for detection of C SFV and PRRSV in food-orientedanimal tissues and clinical samples.

关 键 词：食源性动物组织 猪瘟病毒 猪蓝耳病病毒 双重双色荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]