牛星状病毒和牛诺如病毒双重PCR检测方法的建立及应用  

Establishment and application of a duplex PCR assay for detection of bovine astrovirus and bovine norovirus

作　　者：王文佳[1] 邓廷贤[3] 徐照学[2] 张彬[2] 孟红丽[2] 王亚州[2] 兰亚莉[2] 师志海[2] WANGWen-jia;DENG Ting-xian;XU Zhao-xue;ZHANG Bin;MENG Hong-li;WANG Ya-zhou;LAN Ya-li;SHI Zhi-hai(College of Veterinary Medicine and Pharmaceutical Engineering,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Institute of Animal Husbandry and Veterinary Medicine,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Buffalo Research Institute,Chinese Academy of Agricultural Sciences,Nanning 530001,China)

机构地区：[1]河南牧业经济学院动物医药学院,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]中国农业科学院水牛研究所,广西南宁530001

出　　处：《中国预防兽医学报》2020年第8期786-790,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省重点研发与推广专项(科技攻关)(192102110074);“十三五”国家重点研发计划(2018YFD051700);广西水牛遗传繁育重点试验室开放课题(SNKF-2017-02);国家肉牛牦牛产业技术体系(CARS-37);河南省农业科学院自主创新专项基金(2017ZC51、2018ZC56)。

摘　　要：为建立能同时快速检测牛星状病毒(BAstV)和牛诺如病毒(BNoV)的方法,本研究根据BAstV ORF1a基因和BNoV RdRp基因设计了2对特异性引物,通过优化反应条件,建立了能够同时检测BAstV和BNoV的双重PCR方法。特异性试验结果显示,该方法对牛轮状病毒等5种犊牛腹泻常见病原的扩增结果均为阴性,仅BAstV和BNoV扩增结果为阳性,特异性较强;敏感性试验结果显示,该方法对BAstV和BNoV重组质粒的最低检测限分别为1.09×10^3拷贝/μL和1.42×10^3拷贝/μL,敏感性较高;批内和批间试验结果一致,该方法重复性好。利用该方法和各病原单一PCR方法同时对221份河南省犊牛腹泻粪便样品进行检测,BAstV和BNoV的阳性检出率分别为18.1%和9.96%,两种方法符合率为100%。本研究在国内首次建立了检测BAstV和BNoV的双重PCR方法,其特异性强,敏感性高,重复性好,为BAstV和BNoV的鉴别检测及其分子流行病学调查提供了技术支持。To establish a rapid PCR method for simultaneous detection of bovine astrovirus(BAstV) and bovine norovirus(BNoV), two pairs of specific primers were designed based on ORF1a gene of BAstV and RdRp gene of BNoV. A duplex PCR method was developed after the optimization of amplification conditions. The specific amplification results showed that only BAstV and BNoV were positive, while five other common pathogens causing calf diarrhea were negative. The minimum-detection limits of the method were 1.09×10^3 copies/μL for BAstV plasmid standard and 1.42×10^3 copies/μL for BNoV plasmid standard, respectively.The inter-batch and intra-batch results showed that the method was reproducible. A total of 221 clinical samples, collected from the diarrheic calves in Henan province, were detected by using the duplex PCR method and single PCR method. The positive rates were 18.1% for BAstV and 9.96% for BNoV, respectively. The overall coincidence rate of the two methods was 100%. In this study, a duplex PCR method for detection of BAstV and BNoV was established for the first time in China. These results indicated that the duplex PCR method has good specificity, sensitivity and repeatability, which can be applied widely in differential diagnosis and epidemiological investigation of BAstV and BNoV infection.

关 键 词：牛星状病毒 牛诺如病毒 犊牛腹泻 双重PCR 

分 类 号：S852.65[农业科学—基础兽医学]