荧光环介导逆转录等温扩增(RT-LAMP)技术在病毒性出血性败血症(VHS)诊断中的应用  

Application of Fluorescent Real-time Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Haemorrhagic Septicaemia

作　　者：安元龙[1] 吴斌[2] 林长军[2] 肇慧君[2] 

机构地区：[1]大连工业大学,辽宁大连116034 [2]辽宁出入境检验检疫局,辽宁大连116001

出　　处：《中国动物检疫》2012年第12期23-25,38,共4页China Animal Health Inspection

基　　金：国家质量监督检验检疫总局科技项目(2010IK003)

摘　　要：运用GenieII实时荧光等温扩增检测系统,以LAMP法荧光检测为基础,建立了对病毒性出血性败血症病毒进行荧光实时反转录环介导等温扩增检测方法。根据对VHSV病毒N基因序列的分析,针对病毒的高保守区域的6个特异性序列,设计了3对特异性引物,同时加入dsDNA混合染料,对核酸扩增过程进行实时荧光监控。该方法的特异性良好,与其他鱼类弹状病毒无交叉反应。敏感度高,检测限为20fgRNA模板。实验结果表明该方法是一种操作简单、快速高效、高特异性高灵敏度的病毒检测方法。Using GenieⅡ real-time fluorescence isothermal amplification detection system and based on LAMP fluorescent dection, a fluorescent real-time reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was established for detection of haemorrhagic septicaemia. According to highly conserved regions of nucleoprotein gene of VHSV, three specific primers were designed and, the nucleic acid amplification process was monitored in real-time fluorescent by adding the dsDNA mixed dye. This method was of high specificity, and could not amplify other rhabdoviruses of fish. The detection limit of this assay was 20fg VHSV RNA. The experiment results showed that this method was simple in operation, characterised by rapidity and efficiency, high specificity and sensitivity.

关 键 词：病毒性出血性败血症病毒 荧光逆转录LAMP法 诊断 

分 类 号：S941.413[农业科学—水产养殖]