In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal...In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.展开更多
2010年4月至11月,江苏等地鸭、鹅发生了一种以产蛋率、采食量显著下降,出现脑炎样神经症状为特征的传染病。对发病鹅进行剖检观察,鸭胚尿囊腔途径接种发病鹅病料分离病原,电镜观察病毒粒子。对健康鹅接种分离病毒进行动物回归试验。在...2010年4月至11月,江苏等地鸭、鹅发生了一种以产蛋率、采食量显著下降,出现脑炎样神经症状为特征的传染病。对发病鹅进行剖检观察,鸭胚尿囊腔途径接种发病鹅病料分离病原,电镜观察病毒粒子。对健康鹅接种分离病毒进行动物回归试验。在病毒核酸背景未知的情况下,应用非序列依赖性单引物扩增法结合DNA酶处理（DNase-sequence independent single primer amplification,DNase-SISPA）对病原基因进行扩增,并在此基础上设计了1对特异性引物对病毒基因进行PCR扩增。剖检结果发现病鹅的脑膜、肺脏、肝脏、心脏、卵巢等多器官出血,脾脏肿大坏死。含毒鸭胚尿囊膜超薄切片电镜观察显示：病毒粒子直径为50~60 nm。动物回归试验成功复制出该病并分离到病毒。应用DNase-SISPA方法发现了3个病毒相关基因片段,经序列比对分析,与黄病毒属（Flavi-virus）坦布苏病毒（Tembusu virus）基因序列有很高的同源性,分别为96%、88%、93%。据此设计特异性引物扩增出一段985 bp基因片段,该片段与黄病毒属坦布苏病毒E基因的核苷酸同源性为91%,氨基酸同源性为97%。将分离的鹅黄病毒毒株命名为Goose/Jiangsu/804/2010（简称JS804）。研究结果表明：此次鸭、鹅新发疫病的病原为一种新的黄病毒。展开更多
基金supported by the National Natural Science Foundation of China (31172345)the Jiangsu Provincial Agricultural Science and Technology InnovationFoundation, China (cx(11)4039)
文摘In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.
文摘2010年4月至11月,江苏等地鸭、鹅发生了一种以产蛋率、采食量显著下降,出现脑炎样神经症状为特征的传染病。对发病鹅进行剖检观察,鸭胚尿囊腔途径接种发病鹅病料分离病原,电镜观察病毒粒子。对健康鹅接种分离病毒进行动物回归试验。在病毒核酸背景未知的情况下,应用非序列依赖性单引物扩增法结合DNA酶处理（DNase-sequence independent single primer amplification,DNase-SISPA）对病原基因进行扩增,并在此基础上设计了1对特异性引物对病毒基因进行PCR扩增。剖检结果发现病鹅的脑膜、肺脏、肝脏、心脏、卵巢等多器官出血,脾脏肿大坏死。含毒鸭胚尿囊膜超薄切片电镜观察显示：病毒粒子直径为50~60 nm。动物回归试验成功复制出该病并分离到病毒。应用DNase-SISPA方法发现了3个病毒相关基因片段,经序列比对分析,与黄病毒属（Flavi-virus）坦布苏病毒（Tembusu virus）基因序列有很高的同源性,分别为96%、88%、93%。据此设计特异性引物扩增出一段985 bp基因片段,该片段与黄病毒属坦布苏病毒E基因的核苷酸同源性为91%,氨基酸同源性为97%。将分离的鹅黄病毒毒株命名为Goose/Jiangsu/804/2010（简称JS804）。研究结果表明：此次鸭、鹅新发疫病的病原为一种新的黄病毒。
