| 基于病毒mRNA监测的传染性脾肾坏死病毒灭活快速检验方法建立及其应用 |
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| 引用本文： | 张醴溪,杨圆圆,方伟,付小哲,林强,李趁,刘礼辉,梁红茹,黄志斌,吴志新,李宁求.基于病毒mRNA监测的传染性脾肾坏死病毒灭活快速检验方法建立及其应用[J].水产学报,2017,41(9):1464-1472. |
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| 作者姓名： | 张醴溪 杨圆圆 方伟 付小哲 林强 李趁 刘礼辉 梁红茹 黄志斌 吴志新 李宁求 |
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| 作者单位： | 1. 华中农业大学水产学院,湖北武汉,430070;2. 广东省水生动物疫病预防控制中心,广东广州,510222;3. 中国水产科学研究院珠江水产研究所,农业部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380;淡水水产健康养殖湖北省协同创新中心,湖北武汉430070;4. 中国水产科学研究院珠江水产研究所,农业部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380;5. 华中农业大学水产学院,湖北武汉430070;淡水水产健康养殖湖北省协同创新中心,湖北武汉430070 |
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| 基金项目： | 国家科技支撑计划（2012BAD25B02）；广东省海洋渔业科技推广专项（A201501B12） |
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| 摘 要： | 为建立传染性脾肾坏死病毒(ISKNV)灭活快速检验方法,从ISKNV感染CPB细胞系转录谱筛选并经q RT-PCR验证表达量最高的病毒ORF099基因作为快速检测靶基因。以质粒p MDORF099为标准品,采用q PCR方法绘制了CT值与质粒拷贝数的标准曲线,其线性方程为CT=–3.42lgx+39.455,最低检测限为3拷贝/μL,结果显示组间和组内变异系数均小于2%,表明该方法具有较高的灵敏度和较好的重复性。将ISKNV病毒悬液10倍稀释成100~103拷贝/m L,分别取1 m L病毒稀释液接种CPB细胞,在第7、9和11天提取细胞总RNA,经基因组DNA去除试剂盒去除残留DNA后采用qRT-PCR方法检测ORF099基因转录本,结果显示在第7天即可从接种1个拷贝病毒的细胞中检测出ISKNV ORF099转录本。将3个浓度梯度(0.05%、0.1%、0.2%)的甲醛灭活ISKNV制备的模拟样品以及实验室制备的3批次ISKNV细胞灭活疫苗样品接种CPB细胞9 d,采用上述快速检验方法进行检测。0.05%、0.1%甲醛灭活模拟样品可检测到ISKNV ORF099基因转录本,其他样品均未检测到。而细胞盲传实验显示,0.1%终浓度甲醛灭活ISKNV接种细胞盲传3代未出现CPE,鱼体安全实验显示接种鱼体后无临床发病症状和实验鱼死亡,表明本研究建立的病毒灭活快速检验方法比细胞盲传法和鱼体安全实验具有更高的灵敏度,与传统检测方法相比具有灵敏度高、耗时短、检测效率高等优点,对提高ISKNV灭活疫苗生产效率具有重要意义。
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| 关 键 词： | 传染性脾肾坏死病毒 灭活疫苗 灭活检验 mRNA 荧光定量RT-PCR |
| 收稿时间： | 2016/8/25 0:00:00 |
| 修稿时间： | 2017/1/11 0:00:00 |
Establishment and application of a rapid assay for inactivated infectious spleen and kidney necrosis virus based on viral mRNA |
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| ZHANG Lixi,YANG Yuanyuan,FANG Wei,FU Xiaozhe,LIN Qiang,LI Chen,LIU Lihui,LIANG Hongru,HUANG Zhibin,WU Zhixin and LI Ningqiu.Establishment and application of a rapid assay for inactivated infectious spleen and kidney necrosis virus based on viral mRNA[J].Journal of Fisheries of China,2017,41(9):1464-1472. |
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| Authors: | ZHANG Lixi YANG Yuanyuan FANG Wei FU Xiaozhe LIN Qiang LI Chen LIU Lihui LIANG Hongru HUANG Zhibin WU Zhixin and LI Ningqiu |
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| Institution: | College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China,Guangdong Provincial Aquatic Animal Epidemic Disease Prevention and Control Center, Guangzhou, 510222, China,Guangdong Provincial Aquatic Animal Epidemic Disease Prevention and Control Center, Guangzhou, 510222, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China,Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China,College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China and Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl Piver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510380, China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, 430070, China |
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| Abstract: | |
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| Keywords: | infectious spleen and kidney necrosis virus inactivated vaccine inactivation test mRNA Real-time RT-PCR |
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