鱼类传染性造血器官坏死病毒RT-LAMP检测方法的建立及应用  

Development and application of reverse transcription loop-mediated isothermal amplification for detection of IHNV

作　　者：刘淼[1] 徐黎明[1] 卢彤岩[1] 赵景壮[1] 曹永生[1] 刘红柏[1] 尹家胜[1] 张金凤[1] 冯剑 

机构地区：[1]中国水产科学研究院黑龙江水产研究所 [2]本溪艾格莫林实业有限公司

出　　处：《中国水产科学》2014年第5期1065-1071,共7页Journal of Fishery Sciences of China

基　　金：国家科技支撑计划项目(2012BAD25B10);中央级公益性科研院所基本科研业务费专项(HSY201204)

摘　　要：将逆转录环介导的等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)引入到鱼类的传染性造血器官坏死病病毒(infectious haematopoietic necrosis virus,IHNV)检测中,建立了简单、快速、灵敏的IHNV检测方法。针对IHNV的聚合酶蛋白基因(polymerase protein,L)设计特异性引物,以IHNV基因组RNA为模板,在反转录酶和Bst DNA聚合酶的作用下进行RT-LAMP,反应产物添加SYBR Green I荧光染料后,肉眼观察阳性样本表现为荧光绿色,阴性样本表现为红棕色。2%琼脂糖凝胶电泳检测结果与可视化检测结果一致。该方法实现了检测结果的可视化。特异性分析显示该方法不与传染性胰脏坏死病毒(infectious pancreatic necrosis virus,IPNV)及病毒性出血性败血症病毒(viral haemorrhagic septicaemia virus,VHSV)发生交叉反应,并且可检测不低于10 pfu的IHNV RNA。本研究所建立的IHNV RT-LAMP可视化检测方法具有灵敏度高、特异性好、成本低、不依赖任何专门的仪器设备的优点,其检测结果直观可视化,可实现现场高通量快速检测。A reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was developed for detection of infectious haematopoietic necrosis virus （IHNV）. A set of specific primers were designed based on the IHNV L-protein sequence. IHNV RNA was used as a template for RT-LAMP in a reaction mixture containing reverse transcriptase and Bst DNA polymerase. The RT-LAMP products were observed by the naked eye by adding SYBR Green I fluorescent dye. Positive samples exhibited a fluorescent green color whereas negative samples exhibited a reddish brown color. The RT-LAMP assay had no cross reactivity with infectious pancreatic necrosis virus （IPNV） and viral haemorrhagic septicaemia virus （VHSV）. The RT-LAMP assay was highly sensitive, and could detect 〈 10 pfu IHNV RNA. We confirmed these results using 2% agarose gel eleetrophoresis. In summary, we developed a sensitive, specific, low cost, and simple RT-LAMP method for rapid field diagnosis of IHNV. The method can achieve high throughput detection, without relying on any special equipment.

关 键 词：传染性造血器官坏死病毒(IHNV) 聚合酶蛋白 RT-LAMP 可视化检测 

分 类 号：S941[农业科学—水产养殖]