兔出血症病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立  

Development of SYBR Green Ⅰ Real-time PCR Assay for Detection of Rabbit Hemorrhagic Disease Virus

作　　者：于新友 李天芝 王金良[1,2] 沈志强[1,2] 

机构地区：[1]山东绿都生物科技有限公司,滨州256600 [2]山东省滨州畜牧兽医研究院,滨州256600

出　　处：《中国养兔》2014年第4期19-22,共4页Chinese Journal of Rabbit Farming

基　　金：国家十一五科技支撑计划项目(2006BAD06A14-9);山东省自主创新成果转化重大专项计划(2008ZHZX1A1103);山东省自然科学基金(ZR2010CQ012)

摘　　要：为了建立兔出血症病毒(RHDV)快速、敏感的检测方法。本研究利用RT-PCR技术扩增出RHDVVP60基因中201bp的保守序列,并克隆到p MD18-T载体中作为标准品制作标准曲线,建立了RHDV的SYBR Green Ⅰ荧光定量PCR检测方法。该方法检测灵敏度可达6×101拷贝,与兔水疱性口炎病毒和轮状病毒均不发生交叉反应,具有良好的特异性和重复性。结果表明,建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床RHDV的检测。To develop the SYBR Green I real-time quantitative PCR assay for detection of rabbit hemorrhagicdisease virus(RHDV), a 201 bp conservative region from RHDV VP60 gene was amplified by RT-PCR andcloned into p MD18-T vector to construct recombinant plasmid of p MD18-VP60,which was served as templateto establish the standards curve of the SYBR Green I rea1 time PCR.The results showed that the SYBR GreenI real-time PCR was specifically detected RHDV with a limited detection of 6×101copies and no amplificationfor rabbit vesicular stomatitis virus and rota virus. These data suggested that the SYBR Green I real-time PCRcould be applied in clinical diagnosis and epidemiological investigation for RHDV.

关 键 词：兔出血症病毒 VP60基因 SYBR Green Ⅰ 荧光定量PCR 

分 类 号：S858.291[农业科学—临床兽医学]