牛病毒性腹泻病毒和牛轮状病毒TaqMan二重实时荧光RT-PCR检测方法的建立  

Detection of bovine viral diarrhea virus and bovine rotavirus by TaqMan based real-time RT-PCR

作　　者：范晴[1] 谢芝勋[1] 刘加波[1] 庞耀珊[1] 邓显文[1] 谢志勤[1] 谢丽基[1] 彭宜[1] 

机构地区：[1]广西兽医研究所,广西南宁530001

出　　处：《中国兽医学报》2011年第10期1414-1418,共5页Chinese Journal of Veterinary Science

基　　金：国家百千万人才工程人选专项资金资助项目(945200603);广西科技攻关与新产品试制资助项目(桂科合0992033-5)

摘　　要：根据牛病毒性腹泻病毒(BVDV)5′端非编码区和牛轮状病毒(BRV)VP6基因序列,设计特异性引物和探针。通过对引物和探针浓度、Mg2+浓度、dNTP浓度和Taq酶用量以及反应条件等因素的优化筛选,建立了能同时鉴别BVDV和BRV的二重荧光RT-PCR方法。该方法特异性好,与其他病原如CSFV、MB和IBRV不发生交叉反应;敏感性高,能够检测100个BVDV RNA和100个BRV RNA;稳定性好,批内重复和批间重复变异系数小;干扰性试验表明该方法能同时检测2个模板的不同浓度组合。本研究建立的二重荧光RT-PCR方法可用于BVDV和BRV检测,具有特异、敏感、快速、稳定等优点,是BVDV和BRV基础研究、流行病学调查和临床检测的良好工具。Two pairs of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequence of BVDV 5′ untrascription region and BRV VP6.The reaction parameters such as the concentration of two pair of primers,two probes and other conditions were optimized to develop a duplex real-time RT-PCR assay for rapid detection of BVDV and BRV.It was found that the specificity of this assay was high,and be able to detected BVDV and BRV without other any cross-reactions to CSFV,MB and IBRV.The detection limit of the real-time RT-PCR assay was 100 copies of BVDV viral RNA and BRV viral RNA,indicating a good sensitivity of the assay.The coefficients of variation were both low for the intra-assay and inter-assay tests respectively,indicating a good reliability.When different concentration of BVDV and BRV was mixed together,the result was without any interference.All the resuls indicate that this duplex real-time PCR assay is a specific,sensitive,rapid and reproducible method for detection of BVDV and BRV,and is could applied in fundamental research,clinical detection and epidemiological investigation of BVDV and BRV.

关 键 词：牛病毒性腹泻病毒 牛轮状病毒 TaqMan二重荧光RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]