真鲷虹彩病毒LAMP检测方法的建立

Establishment of LAMP for Detection of Red Sea Bream Iridovirus

为建立一种检测真鲷虹彩病毒的环介导等温扩增(LAMP)方法,满足口岸、养殖场对该病的快速监测的需求,本研究比对分析了Gen Bank中公布的真鲷虹彩病毒基因组序列,筛选其主要衣壳蛋白MCP基因保守区序列,进行LAMP引物设计,建立了真鲷虹彩病毒LAMP检测方法。对扩增条件进行优化,确定最佳反应条件为62℃,45 min。灵敏度试验结果显示,该方法最低检测限为100拷贝/μL目的基因。特异性试验结果显示,该方法不与流行性造血器官坏死病毒(EHNV)、蛙病毒3型(FV3)、甲鱼虹彩病毒(STIV)发生交叉反应。结果表明,本研究建立的LAMP方法具有良好的敏感性和特异性。对5份疑似真鲷虹彩病毒感染的临床样品进行检测,发现检测结果与世界动物卫生组织(OIE)推荐的PCR方法检测结果符合率为100%。研究表明,本方法具有灵敏度高、特异性强,以及仪器设备简单、操作便捷、结果直观等优点,非常适合作为初筛手段,应用于养殖场、口岸等一线的疫病监测。

To develop a Loop-mediated isothermal amplification（LAMP）method of Red sea bream iridovirus（RSIV）for rapid monitoring of the disease at port of entry and ？shery,the RSIV genome sequence published in GenBank was compared and analyzed. The conservative region sequence of the main capsid protein MCP gene was screened out,the LAMP primers were designed,and LAMP for detection of RSIV was established. The conditions of ampli？cation were optimized,and the best reaction condition was 62 ℃,45 min. Sensitivity test results showed that the minimum detection limit for this method was 100 copies/μL. Speci？c test results showed that this method did not cross reaction with EHNV,FV3 and STIV,with good sensitivity and speci？city. By detection of 5 clinical samples,results showed that the coincidence rate of the test results with that of the PCR method recommended by OIE was 100%. This method was a rapid,simple,speci？c and sensitive assay with simple instruments and visualization results,which was suitable for being used as a screening method for monitoring diseases at ports and ？sh farms.