牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用  

Establishment and application of EvaGreen real-time PCR assay for detecting bovine astrovirus

作　　者：师志海[1,2] 王文佳 张彬 孟红丽[1] 王亚州[1] 金磊[1] 兰亚莉 徐照学[1,3] SHI Zhihai;WANG Wenjia;ZHANG Bin;MENG Hongli;WANG Yazhou;JIN Lei;LAN Ya-li;XU Zhaoxue(Institute of Animal Husbandry and Veterinary Medicine,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Veterinary Medicine and Pharmaceutical Engineering,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou 450002,China)

机构地区：[1]河南省农业科学院畜牧兽医研究所,河南郑州450002 [2]河南省畜禽繁育与营养调控重点实验室,河南郑州450046 [3]河南牧业经济学院,动物医药学院,河南郑州450002

出　　处：《中国兽医学报》2021年第1期50-55,共6页Chinese Journal of Veterinary Science

基　　金：河南省重点研发与推广专项基金资助项目(科技攻关)(192102110074);国家肉牛牦牛产业技术体系资助项目(CARS-37);“十三五”国家重点研发计划资助项目(2018YFD051700)。

摘　　要：牛星状病毒(BAstV)是我国新发的犊牛腹泻病原,本试验的目的是建立检测BAstV的Real-time PCR方法。根据BAstV流行株的ORF1a基因序列设计引物,通过优化反应条件和体系,成功建立基于EvaGreen检测BAstV的Real-time PCR方法。结果表明,该检测方法的Ct值与标准品模板在1.36×10^(1)~1.36×10^(8)拷贝/μL线性关系良好,相关系数R^(2)=0.999,扩增效率为93.79%;该方法可特异性检出BAstV,对犊牛腹泻其他相关病原呈阴性;最低检测下限为13.6拷贝/μL;批间和批内的变异系数均小于2%,重复性好。对2017年9月至2019年5月采自河南省的221份犊牛腹泻样本进行检测,BAstV的检出率为18.1%(40/221),采样场阳性率为100.0%(14/14)。本试验所建方法灵敏度高、特异性强、稳定性好,为BAstV的检测和流行病学调查提供了有力手段。Bovine astrovirus(BAstV)is an emerging causative agent of calf diarrhea in China,the aim of the study was to establish a real-time PCR assay for detecting BAstV.One pair of primers were designed based on ORF1 a gene of BAstV.The EvaGreen real-time PCR assay was successfully developed after the optimization of amplification conditions.The test results showed that the Ct value showed a good linear relationship with the standard in the range of 1.36×10^(1)-1.36×10^(8)copies/μL and the correlation coefficient R^(2)=0.999,and the amplification efficiency was 93.79%.There is no specific amplification of other common calf diarrhea pathogen,only BAstV were positive.The detection limit of the method was 13.6 copies/μL for BAstV.The inter-assay and the intra-assay coefficient of variation were both less than 2%,indicating a good repeatability.221 clinical samples collected from the diarrheic calves in Henan Province were detected using this real-time PCR assay,and the BAstV detection rate was 18.1%(40/221),the farms positive rate was 100%(14/14).These results indicated that the real-time PCR assay has good sensitivity,specificity and repeatability,which can be provide an effective means for detection and epidemiological investigation of BAstV.

关 键 词：牛星状病毒 ORF1a基因 实时荧光定量PCR 犊牛腹泻 

分 类 号：S852.65[农业科学—基础兽医学]