传染性鲑鱼贫血症病毒实时荧光环介导等温扩增检测方法的建立

摘要

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。%The infectious salmon anaemia virus (ISAV) is classified as an Orthomyxoviridae. Its genome consists of 8 single-stranded negative-sense RNA segments. ISAV is the pathogen of fatal ISA listed by the World Organization for Animal Health (OIE). It mainly affects salmon farming in Europe and Northern America, but there has been a high chance of its introduction into China due to the increased salmon importation. Therefore it is very important to establish a rapid and accurate method for ISAV detection. Conventional ISAV detection methods involve cell isolation followed by RT-PCR or real-time RT-PCR. Recently Japanese scientists have established a novel technique with high sensitivity and rapidity, namely Loop-mediated isothermal amplification (LAMP) assay. In this study, LAMP assay was developed for detecting infectious salmon anaemia virus (ISAV). Six specific primers were designed according to ISAV genes using LAMP Designer software. A novel LAMP instrument was applied for the amplification and detection. The reaction time and temperature were optimized, and the sensitivity and specificity of this method were analyzed and compared to those of RT-PCR and real-time RT-PCR. The results demonstrated that the optimal amplification temperature of LAMP assay was 64℃, and its detection limit for ISAV RNA was approximately 78.4 fg, which was 100-fold lower than that of traditional RT-PCR but similar to real-time RT-PCR. LAMP assay showed no cross reaction with 14 other fish viruses such as infectious pancreatic necrosis virus (IPNV), spring viraemia of carp virus (SVCV), viral haemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNNV), and yellowtail ascites virus (YAV). This indicated that the primers were highly specific for ISAV. In this study we developed a LAMP assay for ISAV detection. Using specific LAMP assay equipment we could improve the sensitivity and monitor the amplification process. Furthermore this assay does not involve lid opening, hence it greatly reduces the risk of cross contamination.