| 检测绵羊源爱知病毒D型荧光RT-PCR方法的建立和应用 |
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| 引用本文： | 阿比克哈莫,余忠华,杨晨,景志忠,汤承.检测绵羊源爱知病毒D型荧光RT-PCR方法的建立和应用[J].畜牧兽医学报,2022,53(5):1632-1637. |
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| 作者姓名： | 阿比克哈莫 余忠华 杨晨 景志忠 汤承 |
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| 作者单位： | 1. 中国农业科学院兰州兽医研究所 家畜疫病病原生物学国家重点实验室 农业部兽医公共卫生重点实验室, 兰州 730046;2. 阿坝藏族羌族自治州动物科学技术研究所, 红原 624400;3. 西南民族大学畜牧兽医学院, 成都 610041 |
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| 基金项目： | “十三五”国家重点研发计划(2016YFD0500907)； |
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| 摘 要： | 绵羊源爱知病毒D型(ovine Aichivirus D)是在国内绵羊中新发现的病毒，本研究根据绵羊源Aichivirus D 3D基因序列设计检测引物，通过反应体系和条件优化，成功建立了检测绵羊源Aichivirus D的TB Green染料法荧光RT-PCR方法，该方法特异性和稳定性良好，灵敏度高。对2020年4月―2021年5月采集自四川6个场253份绵羊粪便样本(健康羊的133份，腹泻羊的120份)进行检测，结果该病毒的平均检出率为8.7％，场阳性率为66.7％。其中，腹泻粪便样本中绵羊源Aichivirus D阳性率(17.5%)显著高于非腹泻粪便样本中绵羊源Aichivirus D阳性率(0.75%，P<0.001)。表明绵羊源Aichivirus D可能是引起以上地区绵羊腹泻的病原。从绵羊源Aichivirus D的阳性样本中成功克隆出大小为1 422 bp的13个完整的绵羊源Aichivirus D 3D基因，其核苷酸相似性为99.4％～100.0％。遗传演化分析发现这13株绵羊源Aichivirus D 3D基因与本实验室前期研究上传的绵羊源Aichivirus D 3D基因共同聚为单独的一个大支。本研究为绵羊源Aichivirus D的分子检测提供了一种新的方法和基础流行病学数据。
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| 关 键 词： | 绵羊源爱知病毒D型 荧光RT-PCR方法 绵羊 腹泻 |
| 收稿时间： | 2021-08-02 |
Establishment and Application of a Fluorescent RT-PCR Assay for Detecting Ovine Aichivirus D |
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| ABI-Kehamo,YU Zhonghua,YANG Chen,JING Zhizhong,TANG Cheng.Establishment and Application of a Fluorescent RT-PCR Assay for Detecting Ovine Aichivirus D[J].Acta Veterinaria et Zootechnica Sinica,2022,53(5):1632-1637. |
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| Authors: | ABI-Kehamo YU Zhonghua YANG Chen JING Zhizhong TANG Cheng |
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| Institution: | 1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of Ministry of Agriculture and Rural Affairs, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;2. Institute of Animal Science and Technology of Aba Tibetan and Qiang Autonomous Prefecture, Hongyuan 624400, China;3. College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, China |
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| Abstract: | Ovine Aichivirus D is an emerging virus in sheep in China, the aim of this study was to design primers targeting 3D gene of ovine Aichivirus D and optimize the reaction system and conditions, a TB Green Fluorescent RT-PCR method was successfully established; the assay has characteristics of good specificity and stability, high sensitivity. Total 253 sheep fecal samples (133 non-diarrheic and 120 diarrheic) were collected from 6 sheep farms in Sichuan province from April 2020 to May 2021, this virus detection rate was 8.7%, and the farm positive rate was 66.7%(4/6). Notably, the ovine Aichivirus D positive rate for the diarrheic feces (17.5%) was significantly higher than that for non-diarrheic feces (0.75%, P<0.001), suggesting that ovine Aichivirus D may be a pathogen of sheep diarrhea. In addition, 13 complete 3D genes in 1 422 bp in length were successfully cloned from ovine Aichivirus D positive samples in this study, which shared 99.4%-100.0% nt homology with each other. Evolutionary analysis showed that these 13 3D genes together with the ovine Aichivirus D 3D gene uploaded by our previous research clustered into a large independent branch. This study provides a new molecular tool for detecting ovine Aichivirus D and basic epidemiological data of ovine Aichivirus D. |
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| Keywords: | ovine Aichivirus D fluorescent RT-PCR method sheep diarrhea |
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