病毒性出血败血症病毒实时荧光RT-RPA恒温检测方法的建立

Development of a RT-RPA assay for rapid detection of viral hemorrhagic septicemia virus

为建立一种简单、快速的病毒性出血败血症病毒(VHSV)检测方法,本研究通过比较分析VHSV N基因的保守序列,设计了多对引物和一条探针。经过筛选优化,建立了实时荧光RT-RPA检测方法。该方法在39℃恒温反应20 min即可快速、特异性地检测出VHSV,与其他水生动物病毒不发生交叉反应,该方法检测限为1.83×10^3PFU/mL,利用本研究方法对30份鲑鳟鱼临床样品以及9份鱼类疫病病原样品进行检测,检测结果与荧光定量PCR方法结果一致。上述结果表明,本研究建立的检测方法操作简单,反应灵敏,结果可靠,仪器依赖性低,适用于现场对VHSV快速检测。

To establish a rapid and simple method for the detection of viral hemorrhagic septicemia virus(VHSV),a real-time recombinase polymerase amplification(RPA)assay was developed with the multiple pairs of primers and probe designed according to the conserved sequence of VHSV N gene.Through screening the primers and optimizing experimental conditions,the assay can be completed within 20 min at 39℃.VHSV detected quickly and specifically,and has no cross reaction with other aquatic animal viruses.The minimum detected concentration of VHSV was 1.83×10^3 PFU/m L.In addition,30 clinical samples and 9 pathogenic samples of fish diseases were detected by this method,and the detection results were consistent with the real-time PCR.The real-time RPA assay developed in this study was a simple,rapid,sensitive,reliable and affordable method which could potentially be applied for the detection of VHSV in the research laboratory and on site diagnosis.