【摘要】 根据GenBank中鸭瘟病毒UL6和UL7基因的序列,设计了一对特异性引物及TaqMan探针,扩增位于UL6和UL7基因第891￣991位长度为101bp片段。以DPV标准强株DNA为模板,建立了实时荧光定量PCR检测鸭瘟病毒的方法。该方法只能在鸭瘟病毒DNA样本中检出荧光信号,最小检出量为1.57×102copies(23.7fg);线性范围达8个数量级;平行复管检测,组内变异系数为0.84%(n=20),同一组样本(n=6)间隔30d重复检测,结果无显著性差异;对试验感染鸭肝脏和脑组织中鸭瘟病毒的检出率为100%(40/40),对临床病例的检测结果与病毒分离鉴定结果一致;从核酸提取到报告检测结果仅需3h;对鸭正常组织、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、鸡源大肠杆菌、鸭源多杀性巴氏杆菌、鸭副伤寒沙门氏菌等非鸭瘟病毒DNA检测,不出现非特异性荧光信号。更多还原

【Abstract】 According to the UL6 and UL7 gene sequence available in GenBank,the specific primers and TaqMan probe were designed by using Beacon Designer software.The primers amplified a 101 bp fragment between the sites 891-991.With DNA obtained from standard virulent strain of DPV,a real-time quantitative PCR for DPV was established successfully.The results showed this assay was specific and sensitive for DPV with a detection limit of 1.57×102 copies(23.7 fg) and a good linear range(8 log dynamic rang).The coefficient of variation(CV) of intra-assay for same DPV DNA sample was 0.84 %(n=20).The six standard plasmids diluted serially were detected twice with 1 month interval,and the data from two experiments was not significant statistically.The liver and brain samples obtained from ducks experimentally infected with virulent DPV were detected using the assay and all were positive(40/40).For clinical sample detection,the results of real-time PCR assay accorded with the results of isolation and identification of the virus.The whole process would be completed in 3 hours by using this assay.The assay also proven to be specific based on the results that no amplification was found by detecting DNA from normal duck cells,avian infectious laryngotracheitis virus,duck hepatitis virus,E.coli,Pasteurella multocida,S.gallinarum.更多还原