猪A组轮状病毒实时荧光定量PCR检测方法的建立  

Development of Real Time Fluorescent Quantitative PCR Assay for Detection of Porcine Rotavirus A

作　　者：高婉俊[1] 王静[1] 林鸷[1] 曹伟伟[1] 张彦明[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2012年第12期47-53,共7页Progress In Veterinary Medicine

基　　金：教育部高等学校博士点基金项目(博导类)(20110204110015)

摘　　要：本试验旨在建立一种检测猪A组轮状病毒的SYBR GreenⅠ实时荧光定量PCR方法。参考GenBank上登录的猪轮状病毒OSU株的VP6基因序列保守区设计一对特异性引物,通过RT-PCR方法克隆目的基因,并将其连入pMD 19-T Vector,制备阳性标准品,优化反应条件。建立的方法在1.0×102～1.0×109拷贝/μL范围内线性关系良好,反应扩增效率大于90%,扩增相关系数大于0.98。对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒及猪圆环病毒2型均检测不到荧光信号,表明该方法特异性强。该方法批内和批间变异系数均小于2%,重复性好。结果表明,建立的SYBR GreenⅠ实时荧光定量PCR方法可为A组猪轮状病毒早期感染的诊断及病毒定量分析提供技术支持。This study was to establish a SYBR Green I real-time PCR assay for detection of porcine rota- virus. A pair of specific primers targeting the VP6 gene conservative region of porcine rotavirus OSU were designed, the target gene was cloned into the pMD 19-T Vector. A series of diluted the recombinant plasraids containing vp6 gene were used to generate standard curve and the real-time PCR assay was developed after optimizing all kinds of the conditions. A real-time reverse-transcription polymerase chain reaction （RT-PCR） based on SYBR Green ] fluorescent was developed for the quantization of porcine rotavirus. The standard curve generated had a wide dynamic range from 1.0 × 10^2 to 1.0× 10^9 copies/μL, the amplifi- cation efficacy was more than 90%, and the linear correlation was more than 0. 98. No reaction to the cD- NA samples, including classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus and porcine circovirus type 2, was observed. Good reproducibility was obtained because the intra-coefficient variation and inter-coefficient variation were less than 2 0%. The real-time RT-PCR method established in this study should be useful for earlier rapid laboratory diagnosis and quantitative analysis of the infection of the porcine rotavirus A.

关 键 词：A组轮状病毒 SYBR GreenⅠ 实时荧光定量PCR 猪 

分 类 号：S852.659.4[农业科学—基础兽医学] Q789[农业科学—兽医学]