鸭细小病毒地高辛标记探针的制备与应用  

Preparation and application of digoxigenin-labeled nucleic acid probe for detection of duck parvovirus

作　　者：郑肖强 陈浩[1] 窦砚国 杨晶[1] 牛晓宇[1] 于相龙[1] 刁有祥[1] ZHENG Xiao-qiang CHEN Hao DOU Yan-guo YANG Jing NIU Xiao-yu YU Xiang-long DIAO You-xiang~(College of Animal Science and Technology, Shandong Agricultural University, Tai＇an ,Shandong 271018 ,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2016年第12期2001-2004,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31272583;31472199);现代农业产业技术体系建设专项资金资助项目(CARS-43-10);山东省科技发展计划资助项目(2014GNC111023)

摘　　要：根据GenBank发表的1株鸭细小病毒全基因组序列设计1对特异性引物,利用PCR扩增得到大小为301bp的片段,回收纯化后用地高辛进行标记,制备鸭细小病毒地高辛标记探针。特异性试验表明,该探针仅与鸭细小病毒发生特异性杂交反应,与GPV、MDPV、DcuV、DEV、DHAV-I、H9N2亚型AIV、N-DRV、NDV、TMUV杂交反应均为阴性。敏感性试验表明,该探针对鸭细小病毒核酸的最低检测量为25pg。应用制备的探针对山东、江苏和安徽等地采集的61份患病鸭和30份健康鸭组织进行检测,阳性率分别为97%和0%,与病毒分离结果符合率为98%。上述结果显示,本研究制备的探针特异性强、敏感性高,为该病的诊断和流行病学调查提供可靠的方法。According to the genomic sequences of duck parvovirus （DPV） published in GenBank,a pair of primers were designed for amplifying a fragment of 301 bp in length. The amplified product was purified by gel,labeled with digoxigenin（DIG） as DNA probe for detection of duck parvovir- us. Specificity test showed that this probe can develop specificity nuc!eaty hybridization with duck parvovirus, but not with goose parvovirus （GPV）, muscovy duck parvovirus （MDPV）, duck circo- virus （DcuV）, duck enteritis virus （DEV）, cluck hepatitis A virus （DHAV-I）, avian influenza virus （AIV） subtype H9N2, novel duck reovirus（N-DRV）, Newcastle disease virus（NDV）, Tembusu vi- rus（TMUV）. The sensitivity test showed that as low as 25 pg of a DPV＇s DNA could be detected by DIG-labeled probe. Sixty-one samples of diseased ducks with short beak and 30 samples of healthy ducks collected from five flocks of Shandong,Jiangsu and Hebei provinces were used for clinical detection of duck parvovirus using dot blot hybridization. The coincidence rate between the established method and virus isolation was 98 %. These results suggested that the DIG-labeled nu- cleic acid probe was high sensitivity, strong specificity for DPV detection. This method could be used for large-scale diagnosis and epidemiological survey of duck parvovirus infection.

关 键 词：鸭细小病毒 地高辛 探针 

分 类 号：S852.65[农业科学—基础兽医学]