实时荧光定量PCR快速检测鸭病毒性肠炎标准方法的建立

Establishment a standard real-time PCR for rapid detection of duck virus enteritis

根据GenBank（登录号：EF417996）中鸭病毒性肠炎病毒U31基因的序列，设计了1对特异性引物及TaqMan探针，扩增片段长度为76bp。以鸭病毒性肠炎弱毒疫苗株DNA为阳性标准品模板，建立了实时荧光定量PCR检测鸭病毒性肠炎病毒的方法。该方法能在鸭病毒性肠炎病毒DNA样本中检出荧光信号，定量范围为：2．1×10^0～2．1×10^0个拷贝数，最小检出量为2．1×10^0个拷贝；用该方法对人工感染试验的4只鸭组织器官、粪便、血液等样品重复测定3次，病毒模板DNA的检出率为100％，对鸭正常组织、巴氏杆菌、大肠杆菌、沙门菌和鸭病毒性肝炎、鹅源禽流感H5毒株、新城疫、小鹅瘟病毒等DNA检测不出现特异性荧光信号。经过重复性和实际临床样本检验证实，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4h，不仅实现了对鸭病毒性肠炎的快速诊断，也实现了对该病毒DNA由定性到定量的检测。

The specific primers and Taqman probe were designed and synthesized according to the UL31 gene sequence available in GenBank（EF417996）. The fragment is 76 bp. A Taq Real-time fluorescent quantitative PCR assay was established for detection of Duck Plague Virus DNA based on DNA copy obtained from attenuated vaccine strain as a positive template. The sensitivity of the test for DEV is 2.1 ×10^0-2.1 ×10^9 copies. The minimum detection limit is 2.1 copies（2.1 fg）. Tissues,excrement and blood of 4 ducks challenged with DEV were detected repeatly 3 times by this method. All results were positive （3/3）. The specificity of the assay was proven based on no amplification by detecting DNA from normal duck cells, duck viral hepatitis virus, Pasteurella, E. coli, S. gallinarum, Newcastle disease virus,goosling plague virus et al. The whole process of the test could be completed within 4 hours. It brings about quantification detection of DEV DNA.