草鱼呼肠孤病毒873株逆转录环介导等温扩增(RT-LAMP)检测方法研究  

RT-LAMP detection method research for grass carp reovirus strain 873

作　　者：张旻[1] 王娜[1] 景宏丽[1] 吴绍强[1] ZHANG Min;WANG Na;JING Hong-li;WU Shao-qiang(Chinese Academy of Inspection and Quarantine,Beijing 100029,China)

机构地区：[1]中国检验检疫科学研究院,北京100029

出　　处：《淡水渔业》2018年第4期71-75,共5页Freshwater Fisheries

基　　金："十二五"国家科技支撑计划项目(2013BAD12B02)

摘　　要：为提高草鱼呼肠孤病毒(grass carp reovirus,GCRV)的检测效率,根据GCRV-873株VP5基因片段,设计了2对特异性引物,建立了检测GCRV-873株的逆转录环介导等温扩增(RT-LAMP)检测方法。结果显示:该方法使用25μL反应体系,经优化后的反应温度为65℃,反应时间1 h,检测限可达10个拷贝数的病毒核酸,比传统的RT-PCR方法要高10倍。且不与鲤春病毒、传染性造血器官坏死病病毒、传染性胰脏坏死病毒和病毒性出血性败血症病毒RNA产生交叉反应。在反应体系中加入染料后,反应结果肉眼直接可见,是一种特异性强、方便快捷的检测方法,适合GCRV-873株的现场初筛和核酸检测工作。Reverse transcription mediated isothermal amplification（RT-LAMP） detection method of GCRV-873 was established to improve the detection efficiency of grass carp reovirus GCRV.Two pairs of specific primers were designed according to the VP5 gene segment of GCRV strain 873.The results showed that the detection limit reached to 10 copies of viral nucleic acids segment by 25 μL reaction system with optimized reaction temperature of 65 ℃ for 1 h, which was 10 times higher than that of traditional RT-PCR method.The specificity of this assay was high on account of no cross-reactions with RNA detected from spring viraemia of carp virus,infectious haematopoietic necrosis,infectious pancreatic necrosis and Viral Haemorrhagic Septicaemia virus.The results were directly visible to the naked eye after the addition of dyes in the reaction system.In summary,all data confirmed that the RT-LAMP assay was a specific,convenient detection method and suitable for GCRV strain 873 screening and detection.

关 键 词：草鱼呼肠孤病毒 逆转录环介导等温扩增检测方法 检测限 特异性 

分 类 号：S941.4[农业科学—水产养殖]