| 牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用 |
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| 引用本文： | 师志海,王文佳,张彬,孟红丽,王亚州,金磊,兰亚莉,徐照学.牛星状病毒EvaGreen实时荧光定量PCR检测方法的建立及应用[J].中国兽医学报,2021(1):50-55. |
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| 作者姓名： | 师志海 王文佳 张彬 孟红丽 王亚州 金磊 兰亚莉 徐照学 |
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| 作者单位： | ;1.河南省农业科学院畜牧兽医研究所;2.河南省畜禽繁育与营养调控重点实验室;3.河南牧业经济学院动物医药学院 |
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| 基金项目： | 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)。 |
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| 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1.36×101~1.36×108拷贝/μL线性关系良好,相关系数R2=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。
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| 关 键 词： | 牛星状病毒 ORF1a基因 实时荧光定量PCR 犊牛腹泻 |
Establishment and application of EvaGreen real-time PCR assay for detecting bovine astrovirus |
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| SHI Zhihai,WANG Wenjia,ZHANG Bin,MENG Hongli,WANG Yazhou,JIN Lei,LAN Ya-li,XU Zhaoxue.Establishment and application of EvaGreen real-time PCR assay for detecting bovine astrovirus[J].Chinese Journal of Veterinary Science,2021(1):50-55. |
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| Authors: | SHI Zhihai WANG Wenjia ZHANG Bin MENG Hongli WANG Yazhou JIN Lei LAN Ya-li XU Zhaoxue |
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| Institution: | (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) |
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| Abstract: | 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 1.36×101-1.36×108copies/μL and the correlation coefficient R2=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. |
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| Keywords: | bovine astrovirus ORF1a gene real-time PCR calf diarrhea |
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