牛诺如病毒实时荧光定量PCR检测方法的建立及应用  

Establishment and Application of a Real-time PCR Assay for Detecting Bovine Norovirus

作　　者：师志海[1,3] 王文佳 兰亚莉[1] 张彬[1] 孟红丽[1] 王亚州[1] 滑留帅[1] 徐照学[1,3] SHI Zhihai;WANG Wenjia;LAN Yali;ZHANG Bin;MENG Hongli;WANG Yazhou;HUA Liushuai;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,郑州450002 [2]河南牧业经济学院动物医药学院,郑州450046 [3]河南省畜禽繁育与营养调控重点实验室,郑州450002

出　　处：《畜牧兽医学报》2020年第7期1728-1736,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：河南省重点研发与推广专项(192102110074);国家肉牛牦牛产业技术体系(CARS-37);国家重点研发计划(2018YFD051700)。

摘　　要：牛诺如病毒(BNoV)是国内新发的犊牛腹泻病原,笔者拟建立检测BNoV的Real-time PCR方法。根据BNoV流行株的RNA聚合酶(RdRp)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BNoV的Real-time PCR方法。该检测方法的Ct值与标准品模板在2.24×102~2.24×108拷贝·μL-1线性关系良好,相关系数R2=0.997,扩增效率为98.44%;该方法可特异性检出BNoV,对其他犊牛腹泻相关病原呈阴性;最低检测限为22.4拷贝·μL-1;批间和批内的变异系数均小于2%,重复性好。对2017年9月—2019年5月采自河南省的221份犊牛腹泻样本中BNoV的检出率为11.31%(25/221),采样场阳性率为92.86%(13/14)。本试验所建方法灵敏度高、特异性强、稳定性好,为BNoV的检测和流行病学调查提供了有力手段。Bovine norovirus(BNoV)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 BNoV.One pair of primers was designed based on RNA-dependent RNA polymerase(RdRp)gene of BNoV.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 2.24×102-2.24×108copies·μL-1 and the correlation coefficient R2=0.997,and the amplification efficiency was 98.44%.There is no specific amplification of other common calf diarrhea pathogens,only BNoV were positive.The detection limit of the method was 22.4 copies·μL-1 for BNoV.The inter-assay and the intra-assay coefficient of variation were both less than 2%,indicating a good repeatability.221 clinical samples that collected from the diarrheic calves in Henan Province during September 2017 to May 2019 were detected using this real-time PCR assay,and the BNoV detection rate was 11.31%(25/221),the farms positive rate was 92.86%(13/14).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 BNoV.

关 键 词：牛诺如病毒 实时荧光定量PCR 犊牛腹泻 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]