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根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了ISAV的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。
According to the gene conservative sequence of ISAV, six primers were designed by using LAMP Designer software, and amplified by a novel loop-mediated isothermal amplification device. The reaction conditions were optimized and the specificity and sensitivity of the established method were analyzed. Compared with RT-PCR and real-time fluorescence RT-PCR. The results show that the optimal reaction temperature is 64 ℃ and the obvious amplification can be observed after 10 min reaction. The sensitivity of this method was high, with a detection limit of 78.4 fg RNA, which was 100-fold more sensitive than conventional RT-PCR and had the same sensitivity as real-time fluorescence quantitative RT-PCR. The specificity was good with IPNV, 14 major fish viruses such as SVCV, VHSV, VNNV and YAV did not cross-react. The results showed that the real-time fluorescence ring-mediated isothermal amplification (ISAV) detection method of ISAV was established in this study. The experiment can monitor the whole amplification process in real time and improve the sensitivity of detection. At the same time, it can prevent the contamination caused by the open- .