牛冠状病毒、牛诺如病毒和牛嵴病毒多重PCR检测方法的建立及初步应用  

Establishment and preliminary application of a multiple PCR for detection of BCoV,BNoV and BKV

作　　者：王文佳 徐照学[2,3] 兰亚莉 师志海[2,3] WANG Wen-jia;XU Zhao-xue;LAN Ya-li;SHI Zhi-hai(College of Pharmaceutical Engineering,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Institute of Animal Husbandry and Veterinary,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou 450002,China)

机构地区：[1]河南牧业经济学院制药工程学院,河南郑州450046 [2]河南省农业科学院畜牧兽医研究所,河南郑州450002 [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：《中国兽医学报》2020年第7期1306-1310,1355,共6页Chinese Journal of Veterinary Science

基　　金：“十三五”国家重点研发计划基金资助项目(2018YFD0501700);广西水牛遗传繁育重点实验室开放课题基金资助项目(SNKF-2017-02);国家肉牛牦牛产业技术体系基金资助项目(CARS-37);河南省农业科学院自主创新专项基金资助项目(2017ZC51,2018ZC56);河南省“四优四化”科技支撑行动计划基金资助项目(2017-106-2130106)。

摘　　要：为建立能同时检测牛冠状病毒(bovine coronavirus,BCoV)、牛诺如病毒(bovine norovirus,BNoV)和牛嵴病毒(bovine kobuvirus,BKV)的方法,根据BCoV、BNoV和BKV基因组的保守区域设计3对特异性引物,预期特异性扩增BCoV、BNoV和BKV的片段大小分别为896,542,216 bp,建立了BCoV、BNoV和BKV的多重PCR检测方法。特异性检测结果显示,该方法能特异性扩增BCoV、BNoV和BKV的目的条带,且未扩增出其他犊牛腹泻(calf diarrhea,CD)病原;BCoV、BNoV和BKV的最低检测限分别为9.51×10^5,5.39×10^5,2.23×10^6 copies/μL。对采集自河南部分地区的127份CD样品的检测结果显示,BCoV的阳性率为14.96%(19/127),BNoV的阳性率为6.30%(8/127),BKV的阳性率为4.72%(6/127),三者与单一PCR检测结果相一致。结果表明,本研究建立的多重PCR方法可用于BCoV、BNoV和BKV的检测和流行病学调查。To establish a PCR method for simultaneous detection of bovine coronavirus(BCoV),bovine norovirus(BNoV)and bovine kobuvirus(BKV),three pairs of specific primers were designed based on the conserved regions of BCoV,BNoV and BKV genomes.The specific amplification of BCoV,BNoV and BKV were 896,542 and 216 bp,respectively.The method had a sensitivity of 9.51×10^5 copies/μL for BCoV,5.39×10^5 copies/μL for BNoV and 2.23×10^6 copies/μL for BKV,as well as a good specificity,which was only positive for BCoV,BNoV and BKV,but negative for other calf diarrhea virus pathogens.Furthermore,this multiple PCR method was used to test 127 clinical samples that collected from the diarrheic calves in Henan Province.The positive rate was 14.96%(19/127),6.30%(8/127)and 4.72%(6/127)for BCoV,BNoV and BKV,which is consistent with single PCR results.The multiple PCR method can be used for the rapid detection and epidemiological investigation of BCoV,BNoV and BKV.

关 键 词：牛冠状病毒 牛诺如病毒 牛嵴病毒 CD 多重PCR 

分 类 号：S852.65[农业科学—基础兽医学]