牛诺如病毒TaqMan实时荧光定量PCR方法的建立  

Establishment of a TaqMan 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] 徐照学[1,3] SHI Zhi-hai;WANG Wen-jia;LAN Ya-li;ZHANG Bin;MENG Hong-li;WANG Ya-zhou;XU Zhao-xue(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]河南牧业经济学院动物医药学院,河南郑州450046 [3]河南省畜禽繁育与营养调控重点实验室,河南郑州450002

出　　处：《中国兽医科学》2020年第8期969-975,共7页Chinese Veterinary Science

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项目(CARS-37);国家重点研发计划项目(2018YFD051700);广西水牛研究所开放课题项目(SNKF-2017-02)。

摘　　要：牛诺如病毒(BNoV)是国内新发的犊牛腹泻的病原,为建立高效快速、特异的BNoV定量检测方法,本研究参照GenBank登录的BNoV的RdRp基因的保守序列,设计合成了1对特异性引物和1条探针,通过优化反应条件和体系,建立了BNoV的TaqMan荧光定量PCR方法。结果显示,该方法选择的引物具有高度特异性,只对BNoV有特异性扩增,而对牛冠状病毒等4种犊牛腹泻常见病原未见扩增;对BNoV核酸最低检测限为15.9 copies/μL,且批内、批间变异系数均小于2%,重复性好。对采集自2017年9月至2019年11月河南、山东和四川省的261份犊牛腹泻样本进行检测,结果显示BNoV的阳性检出率为11.49%(30/261)。本研究建立的TaqMan实时荧光定量PCR方法对BNoV的病原检测和流行病学调查具有重要意义。Bovine norovirus(BNo V) is an emerging causative agent of calf diarrhea in China.In order to establish an efficient,rapid and specific method for quantitative detection of BNo V infection,one pair of specific primers and probe were designed based on the conserved sequence of Rd Rp gene of BNo V.After optimizing the reaction conditions and reaction system,a Taq Man real-time PCR method for detection of BNo V was successfully established.The results showed that the primers selected by this method were highly specific.It could specifically detect BNo V,no cross-reaction with other four common calf diarrhea pathogens such as bovine rotavirus.The detection limit of BNo V was reach to 15.9 copies/μL,and the coefficient variation of intra-and inter-assay were less than 2%,indicating the established method had high repeatability.The detection of 261 clinical samples collected from the diarrheic calves in Henan Province,Shandong Province and Sichuan Province during September 2017 to November 2019 showed that the positive detection rate of BNo V was 11.49%(30/261).The established TaqMan real-time PCR method could contribute to pathogenic surveillance and epidemiological investigation of BNo V infections in calves.

关 键 词：牛诺如病毒 犊牛腹泻 TaqMan荧光定量PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]