传染性造血器官坏死病病毒RT-RAA-LFD检测方法的建立与应用

Development and application of an RT-RAA-LFD assay for rapiddetection of infectious haematopoietic necrosis virus

为针对传染性造血器官坏死病病毒(IHNV)建立一种可实现现场快速、准确的检测方法,结合重组酶介导扩增(RAA)和侧向流试纸条(LFD)技术,在IHNVG基因保守区域设计引物和探针,通过优化反应时间和温度等条件,建立了可用于IHNV现场可视化检测的RT-RAA-LFD方法。优化后的检测方法在35℃水浴锅中恒温反应15 min即可实现对IHNV目的基因片段的有效扩增。结果显示:该方法可以特异性地检测出IHNV,不与其他常见鱼类病毒发生交叉反应;对IHNV质粒标准品的检测下限可以达到2.15×101 copies/μL;采用该方法和实时荧光RT-PCR、实时荧光RT-RAA同时对35份临床样本进行检测,其阳性符合率为100%。上述结果表明,本研究针对IHNV成功建立了一种快速、准确、便捷的试纸条检测方法,并可适应于多种场合。

To establish a rapid and accurate method for detection of infectious haematopoietic necrosis virus(IHNV),primers and probes were designed according to the conserved sequence of IHNV G gene.A real-time recombinase-aid amplification combined with lateral flow dipstick(RT-RAA-LFD)method was established for visual detection of IHNV in the field with a water bath within 15 min at 35℃after being optimized.The assay was specific with the target sequence of IHNV with a detection limit up to 2.15×101 copies/μL.Thirty-five clinical samples were tested by this method,showing 100%consistency with the real-time RT-PCR and RT-RAA.The RT-RAA-LFD assay developed in this study was a rapid,reliable and simple method which can be broadly used in the farms and diagnostic on the samples with suscepected clinical signs.