| 实时荧光定量RT-PCR检测牛库布病毒 |
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| 引用本文： | 李慧平,汤承,岳华.实时荧光定量RT-PCR检测牛库布病毒[J].畜牧兽医学报,2020,51(4):888-893. |
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| 作者姓名： | 李慧平 汤承 岳华 |
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| 作者单位： | 1. 西南民族大学生命科学与技术学院, 成都 610041;2. 国家现代农业产业技术体系四川省兽药创新团队, 成都 610041 |
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| 基金项目： | 西南民族大学创新团队（2020NTD02）；西南民族大学研究生“创新型科研项目”（CX2018SZ30） |
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| 摘 要： | 牛库布病毒（bovine kobuvirus，BKoV）可能是一个腹泻相关病原，目前还没有检测该病毒的实时荧光定量RT-PCR方法的报道。依据BKoV 3D核苷酸序列，采用Primer 6.0设计了一对引物，通过优化反应条件和反应体系，成功建立了检测BKoV的TB Green染料法实时荧光定量RT-PCR方法。结果显示此方法在4.86×108~4.86×102 copies·μL-1病毒浓度之间具有良好的线性关系，相关系数为0.999 5，扩增效率为92.75%；此方法只特异性检测BKoV，而与其他常见的腹泻病原没有交叉反应；组内及组间的变异系数均小于2%；最低检测下限为4.86×102 copies·μL-1。本研究所建的实时荧光定量RT-PCR方法对奶牛和牦牛腹泻粪便样本中BKoV的检出率明显高于已报道的3篇有关RT-PCR方法。对2018年5月-2019年5月采集自青藏高原地区（青海、西藏和四川藏区）的131份牦牛腹泻粪便样本进行检测，结果显示BKoV检出率为31.30%。首次成功建立了检测BKoV的实时荧光定量RT-PCR方法，具有良好的特异性和稳定性，且灵敏度高，为BKoV的检测及流行病学调查提供了一种新的技术手段；并且首次证实BKoV在牦牛中的流行，为牦牛腹泻的防控问题提供了参考。
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| 关 键 词： | 牛库布病毒 实时荧光定量RT-PCR 牦牛 腹泻 |
| 收稿时间： | 2019-10-21 |
Development and Application of a Real-time RT-PCR Assay for Detecting Bovine Kobuvirus |
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| LI Huiping,TANG Cheng,YUE Hua.Development and Application of a Real-time RT-PCR Assay for Detecting Bovine Kobuvirus[J].Acta Veterinaria et Zootechnica Sinica,2020,51(4):888-893. |
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| Authors: | LI Huiping TANG Cheng YUE Hua |
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| Institution: | 1. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China;2. Sichuan Veterinary Medicine and Drug Innovation Group of China Agricultural Research System, Chengdu 610041, China |
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| Abstract: | Bovine kobuvirus (BKoV) is a potential diarrhea-causing pathogen in calves, and there is no real-time RT-PCR assay for detecting this virus until now. By designing primers targeted to 3D fragment of BKoV strains and optimizing reaction conditions and system, a TB Green real-time RT-PCR assay was successfully developed. The results demonstrated that the assay had a good linear relationship between 4.86×108 and 4.86×102 copies·μL-1 of virus concentration, the linear correlation coefficient was 0.999 5 and the amplification efficiency was 92.75%. This assay was only specific for detection of BKoV, no cross reaction was observed with other species of diarrhea-causing pathogens; the minimum detection limit was 4.86×102 copies·μL-1; both the intra- and inter-group coefficients of variation were <2%. Comparing to other three reported RT-PCR assays, the assay had a significant high detection rate of BKoV for clinical feces samples from both diarrheal cows and yak. Total 131 yak diarrhea samples collected from Qinghai, Xizang and Sichuan region between May 2018 and May 2019, the BKoV detection rate was 31.30%. The real-time RT-PCR assay for detecting BKoV developed in this study has a good specificity and stability, which provide an effective method for detection and epidemiological investigation of BKoV. |
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| Keywords: | bovine kobuvirus real-time RT-PCR yak diarrhea |
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