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鲤春病毒血症病毒(SVCV)、病毒性出血性败血症病毒(VHSV)和传染性造血器官坏死病病毒(IHNV)是严重危害水产养殖业的3种鱼类弹状病毒。为建立这3种鱼类弹状病毒的快速检测方法,本研究设计合成针对这3种病毒系列引物,并在扩增反应体系中加入荧光染料SYBR Green I,通过恒温荧光检测仪Deaou-308C扩增检测,分别建立了这3种病毒的荧光定量环介导等温RNA扩增(RT-LAMP)方法。经反应条件的优化,检测结果显示,这3种方法分别对SVCV、VHSV和IHNV 3种弹状病毒具有特异性的扩增反应,对传染性胰坏死病毒、狗鱼弹状病毒和牙鲆弹状病毒核酸的扩增结果均为阴性,具有良好的特异性。灵敏度试验最低检测限分别为4.0×104拷贝/μL、5.4×104拷贝/μL、4.3×104拷贝/μL。该方法在63℃下保温40 min可以完成检测,操作简单、灵敏度高、特异性强,通过恒温实时荧光检测仪可实时检测及自动判读检测结果,适合现场检测和口岸快速检测。
The SVCV, VHSV and IHNV are three types of fish rhabdovirus that seriously endanger the aquaculture industry. In order to establish a rapid detection method for these three kinds of fish rhabdoviruses, we designed and synthesized these three kinds of viral primers and added the SYBR Green I fluorescent dye to the amplification reaction system. Through the Deaou-308C Increased detection of these three viruses were established fluorescence quantitative ring-mediated isothermal RNA amplification (RT-LAMP) method. The optimization of the reaction conditions showed that these three methods had specific amplification reactions against SVCV, VHSV and IHNV three kinds of rhabdoviruses, respectively. The detection of infectious pancreatic necrosis virus, The results of the amplification of the recombinant virus nucleic acids are negative and have good specificity. Sensitivity test minimum detection limit were 4.0 × 104 copies / μL, 5.4 × 104 copies / μL, 4.3 × 104 copies / μL. The method can be tested at a temperature of 63 ° C for 40 min, which has the advantages of simple operation, high sensitivity and strong specificity, real-time detection and automatic interpretation of the detection results by the real-time fluorescence detector, and is suitable for field detection and rapid port detection.