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以真鲷虹彩病毒(Red-sea breamiridovirus,RSIV)主要衣壳蛋白(Major capsid protein,MCP)的基因保守片段为靶序列,利用Pri mer Express3.0软件设计定量PCR引物,建立了RSIV的SYBR GreenⅠ实时定量PCR检测方法。将RSIV MCP基因连接pMD18-T载体,构建重组质粒,经过梯度稀释后作为标准品,根据标准品拷贝数(X)与Ct值的关系绘制了标准曲线,为Ct=-3.1841gX+40.270,相关系数R2=0.9969。熔解曲线分析表明,定量PCR产物的Tm值为82.5℃。该方法的检测限为2.20×102拷贝/反应,对流行性造血器官坏死病毒、淋巴囊肿病毒、蛙病毒3、甲鱼虹彩病毒都没有扩增反应,具有特异性。利用该方法对84批海水鱼类(石鲽、大菱鲆、鲈鱼)进行检测,其中5批鱼样品感染RSIV,并利用标准曲线对病毒含量进行了定量分析。
The conserved fragment of Major capsid protein (MCP) of Red-sea breamiridovirus (RSIV) was used as target sequence. Primer Express 3.0 software was used to design quantitative PCR primers and SYBR GreenⅠ Real-time quantitative PCR detection method. The RSIV MCP gene was ligated into the pMD18-T vector to construct a recombinant plasmid. After gradient dilution, the standard curve was prepared according to the relationship between the standard copy number (X) and the Ct value, which was Ct = -3.1841gX + 40.270, The coefficient R2 = 0.9969. Melting curve analysis showed that the Tm value of the quantitative PCR product was 82.5 ° C. The detection limit of the method was 2.20 × 102 copies / reaction, which was specific to epidemic hematopoietic necrosis virus, lymphatic cyst virus, frog virus 3 and iridescent mechitis virus. Using this method, 84 batches of marine fish (Scirpus, Turbot, Perch) were tested. Five batches of fish were infected with RSIV and the virus content was quantitatively analyzed by a standard curve.