鸭坦布苏病毒SYBR GreenⅠ实时荧光定量检测方法的建立  

Establishment of a SYBR GreenⅠ-based real-time RT-PCR for rapid detection of duck Tembusu virus

作　　者：万春和[1] 朱海侠[1] 施少华[1] 黄瑜[1] 程龙飞[1] 傅光华[1] 陈红梅[1] 

机构地区：[1]福建省农业科学院畜牧兽医研究所福建省畜禽疫病防治工程技术研究中心,福建福州350013

出　　处：《中国兽医学报》2013年第7期973-978,共6页Chinese Journal of Veterinary Science

基　　金：现代农业产业体系建设专项资金资助项目(CARS-43);公益性行业(农业)科研专项(201003012);福建省种业创新与产业化工程项目(FJZYCX-9);公益类科研院所专项(2011R1025-8);福建省自然科学基金资助项目(2012J001112);福建省农业科学院创新团队项目(STIF-Y02)

摘　　要：根据鸭坦布苏病毒(Duck Tembusu virus,DFV)NS5基因序列特征设计引物,建立基于SYBR GreenⅠ检测模式的实时荧光定量RT-PCR(real-time RT-PCR,RRT-PCR),该方法检测DFV NS5基因2.74×103～2.74×107拷贝/μL反应范围内有很好的线性关系。扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(86.23±0.18)℃,对禽流感病毒、鸭肝炎病毒、鸭源禽1型副黏病毒、鸭减蛋综合征病毒、番鸭呼肠孤病毒核酸均无阳性信号扩增,可重复性好,组内变异系数为0.52%～1.48%,组间变异系数0.71%～2.21%。检测速度快,从样本处理到报告结果仅需4h。A pair of specific primers targeted to non structure gene 5 （NSS） of duck Tembusu virus was designed and a SYBR Green I fluorescent based real-time RT-PCR （RRT-PCR） was devel- oped for the quantization of duck Tembusu virus. The detection limit of RRT-PCR was 2.74 × 102 plasmid copies. The melting curve analysis using SYBR Green I dye showed one specific peak, a melting temperature （Tm） was （86.23±0.18） ℃ ,and no primer-dimers peak was observed. No amplification was detected from unrelated virus samples by this method, such as avian influenza virus, duck hepatitis virus type 1, avian paramyxovirus type 1, egg drop syndrome virus, duck reo- virus. Fine reproducibility was obtained for detecting plasmid DNA with intra-assay of 0. 52%- 1.48G and inter-assay of 0.71G-2.21G. The real-time PCR method developed in this study will be useful for rapid laboratory diagnosis and epidemiology investigation for duck Tembusu virus.

关 键 词：鸭坦布苏病毒 SYBR GreenⅠ 实时荧光定量RT-PCR 

分 类 号：S852.657[农业科学—基础兽医学]