鲤浮肿病毒荧光RAA快速检测方法的建立

Development of a real-time recombinase-aid amplification assay for rapid detection of carp edema virus

为建立一种简单、快速的鲤浮肿病毒(CEV)重组酶介导的链替换扩增(RAA)检测方法,本研究通过比较分析CEVP4a基因序列,设计引物与探针,经过筛选优化,建立了实时荧光RAA检测方法。结果显示,该方法在39℃恒温反应20min即可快速、特异性地检测出CEV,与常见的鱼类病毒不发生交叉反应,其对质粒标准品的检测下限为2.8×10^1拷贝/μL,组内和组间变异系数均小于5%。利用本研究建立的方法对35份临床鲤科鱼类样品进行检测,检测结果与荧光定量PCR方法结果一致。本研究建立的检测方法操作简单,特异性强、敏感性高,结果可靠,不依赖于复杂的仪器,适用于现场对CEV的快速检测。

To establish a rapid and simple method for the detection of carp edema virus(CEV),a real-time recombinase-aid amplification(RAA)assay was developed with the primers and probe designed according to the conserved sequence of CEV P4a gene.Through screening the primers and optimizing experimental conditions,the assay can be completed within 20 min at 39℃.The assay was specific to amplify the target sequence of CEV with a detection limit up to 2.8×10^1 copies per reaction.The co-efficient of variations in intra-and inter-assay were both less than 5%.In addition,35 clinical samples were tested by this method and 7 samples were detected to be positive,which was 100%consistent with the real-time PCR.The real-time RAA assay developed in this study was a simple,rapid,sensitive,reliable and affordable method which could potentially be applied for the detection of CEV in the research laboratory and on site diagnosis.