| 猪丁型冠状病毒荧光定量RT-PCR与S1蛋白间接ELISA检测方法的建立及应用 |
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| 引用本文： | 王经纬,雷喜梅,覃盼,赵鹏伟,王斌,王逸雯,李懿婷,金颢蕊,李龙,黄耀伟.猪丁型冠状病毒荧光定量RT-PCR与S1蛋白间接ELISA检测方法的建立及应用[J].生物工程学报,2017,33(8):1265-1275. |
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| 作者姓名： | 王经纬 雷喜梅 覃盼 赵鹏伟 王斌 王逸雯 李懿婷 金颢蕊 李龙 黄耀伟 |
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| 作者单位： | 1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058,2 杭州贝尔塔生物技术有限公司，浙江 杭州 310004,1 浙江大学 动物科学学院 动物预防医学研究所，浙江 杭州 310058 |
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| 基金项目： | 国家重点研究发展计划 (No. 2016YFD0500102) 资助。 |
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| 摘 要： | 最近,新发现的猪丁型冠状病毒(Porcine deltacoronavirus,PDCo V)可导致仔猪腹泻,亟待建立有效准确的核酸及血清学检测方法并调查猪场的PDCo V感染情况。本研究采用针对病毒M基因的探针荧光定量RT-PCR检测方法,对2014-2015年间收集的河南、湖南、浙江、江西、安徽、河北、黑龙江、江苏、山东和上海等10个省市收集的共254份腹泻仔猪小肠匀浆或粪便样本进行检测,共检出11份PDCo V阳性样本,总阳性率为4.33%。采用在昆虫细胞表达纯化的PDCo V囊膜S1蛋白为包被抗原,建立间接酶联免疫吸附试验(ELISA)方法,检测2015-2016年间收集的来自江苏、湖南、浙江等7个省份的609份具有腹泻症状的猪血清样品,抗PDCo V S1抗体检出率为44.17%(269/609)。上述建立的两种方法分别针对病毒的RNA或抗体对PDCo V进行特异性检测,可以应用于PDCo V流行情况的监控。
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| 关 键 词： | 猪丁型冠状病毒，实时荧光定量RT-PCR，间接ELISA，S1基因 |
| 收稿时间： | 2017/3/25 0:00:00 |
Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus |
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| Jingwei Wang,Ximei Lei,Pan Qin,Pengwei Zhao,Bin Wang,Yiwen Wang,Yiting Li,Haorui Jin,Long Li and Yao-Wei Huang.Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus[J].Chinese Journal of Biotechnology,2017,33(8):1265-1275. |
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| Authors: | Jingwei Wang Ximei Lei Pan Qin Pengwei Zhao Bin Wang Yiwen Wang Yiting Li Haorui Jin Long Li and Yao-Wei Huang |
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| Institution: | 1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China,2 Hangzhou Belta-Biotechnology Co., Ltd., Hangzhou 310004, Zhejiang, China and 1 Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China |
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| Abstract: | Porcine deltacoronavirus (PDCoV) has been recently recognized as an emerging viral pathogen that causes diarrhea in newborn piglets. A total of 254 small intestinal or fecal samples collected from 10 provinces including Henan, Hunan, Zhejiang, Jiangxi, Anhui, Hebei, Heilongjiang, Jiangsu, Shandong and Shanghai between 2014 and 2015, were screened by quantitative RT-PCR targeting the viral M gene. Eleven PDCoV positive samples were identified with a total positive rate of 4.33%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed based on the recombinant S1 protein of PDCoV. This assay was used to test 609 serum samples of pigs with diarrhea symptoms collected from 10 provinces between 2015 and 2016. The positive rate of PDCoV antibody was 44.17% (269/609). The two methods can be used to monitor the PDCoV epidemiology in the levels of PDCoV specific RNA or antibody, helping better prevent and control PDCoV. |
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| Keywords: | porcine deltacoronavirus real-time RT-PCR indirect ELISA S1 gene |
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