鸭黄病毒荧光定量RT-PCR检测方法的建立及应用

Development and application of real-time RT-PCR assay for duck flavivirus

根据鸭黄病毒E蛋白基因序列设计了1对特异性引物和TaqMan探针,建立了实时荧光定量RT-PCR检测鸭黄病毒的方法。根据含鸭黄病毒目的扩增片段的质粒拷贝数与定量反应Ct值的关系,绘制了标准曲线(Y=-3.39 X+43.18,r=0.973)。该方法具有特异性,对鸭病毒性肝炎病毒、禽流感病毒、新城疫病毒、腺病毒、鸭瘟病毒、猪乙脑病毒的核酸都没有扩增反应。敏感性试验显示,建立的此方法最低可检出1.9个TCID50病毒核酸。该方法对5只健康雏鸭人工感染86h后的组织器官样品(盲肠扁桃体除外)的检测结果均为阳性,与病毒分离鉴定的阳性符合率为100%。结果表明,该方法真实可靠,而且从核酸提取到报告检测结果耗时不超过3h,为鸭黄病毒病提供了一种敏感特异的定量检测方法。

A real-time RT-PCR method was developed for the detection of duck flavivirus（DFV）.The specific primers and TaqMan probe were designed and synthesized according to the E gene sequence of duck flavivirus.The standard curve（Y=-3.39 X＋43.18,r=0.973） was plotted based on the linear relationship between the amount of plasmid DNA and cycle threshold（Ct）.This method was specific for duck flavivirus（DFV） but not for duck hepatitis virus,avian influenza virus,Newcastle disease virus,adenovirus,duck plague virus and porcine Japanese encephalitis virus.The detection limit reached 1.9 TCID50 of the virus under the optimized conditions.Tissues in five ducks after 86 h artificial infected with DFV were tested positive by this method,without parts of cecal tonsils.And the positive detection rate of the method was consistent with routine virus isolation method.These results indicated that the real-time RT-PCR approach provides a powerful diagnostic tool with high sensitivity and specificity for the identification and quantitation of DFV and the whole process of the test could be completed within 3 hours.