探针检测鸭黄病毒的地高辛标记DNA的制备与应用

利用RT-PCR方法扩增鸭黄病毒的NS3基因406bp的特异性片段,回收并纯化PCR产物,用地高辛标记,制备核酸探针。特异性试验结果表明,该探针仅与鸭黄病毒的核酸特异性杂交,而与鸭瘟病毒、H9N2禽流感病毒、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒的核酸杂交均为阴性。敏感性试验表明,该探针对鸭黄病毒的RNA最低检出限量为100μg/L。对疑似黄病毒感染鸭的肝脏、肺脏、脾脏、输卵管、卵泡膜和泄殖腔棉拭子进行检测,以卵泡膜的检出率最高。该研究为鸭黄病毒感染的诊断和流行病学调查提供了一种可靠的方法。

Preparation of digoxigenin-labeled DNA probe for detection of duck flavivirus and its application

The 406 bp specificity fragments was amplified by PCR from NS3 gene in flavivirus and through reclamation and cleansing,this specificity fragment was labeled by digoxigenin（DIG）,then a novel DIG-labeled DNA probe method was established.Specificity test showed that this probe can develop specificity nucleate hybridization with flavivirus,but not with duck plagues virus（DPV）,avian influenza virus（AIV） subtype H9,Newcastle disease virus（NDV）,infectious bursal disease virus（IBDV）,egg drop syndrome virus（EDS-76）.As little as 100 μg/L of flavivirus RNA could be detected with the DIG-labeled probe.The detection results for clinical samples demonstrated that the flavivirus-specific RNA was most frequently detected in theca folliculi,followed by the liver,lungs,spleen,oviduct and cloacal swab.These results suggested that the DIG-labeled DNA probe assay could be used as a method for the diagnosis and detection of clinical cases,and for molecular epidemiological investigation of duck flavivirus.