一株新型鸭呼肠孤病毒的分离及抗体检测的间接ELISA建立  

Isolation of a Novel Duck Reovirus Strain and Establishment of Indirect ELISA for Detecting Antibodies

作　　者：徐晨晨 马旭杰 宋素泉[1] 闫丽萍 XU Chenchen;MA Xujie;SONG Suquan;YAN Liping(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2024年第12期5651-5662,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金面上项目(32272994)。

摘　　要：新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)能造成多品种雏鸭肝、脾的出血坏死,并造成免疫抑制,给我国的水禽养殖业造成了严重经济损失,但市场上仍无商品化检测试剂盒。基于此,本研究通过蚀斑纯化从临床病料中成功分离到一株纯净的新型鸭呼肠孤病毒(命名为E232株),并以原核表达的σC蛋白为包被原建立了能够检测NDRV血清抗体的间接ELISA。遗传进化分析结果显示,E232株属于NDRV,且与其他NDRV毒株的核苷酸同源性为96.4%~98.9%。动物回归试验结果表明,E232株发病率达到100%,发病雏鸭的脾出现严重肿胀、出血、坏死,泄殖腔排毒量和咽部排毒量均在感染后第5天达到高峰。建立的间接ELISA具有良好的特异性,与其他鸭病病原阳性血清均无交叉反应;检测NDRV阳性血清最低检出效价为1∶6400,批内重复变异系数为1.53%~7.24%,批间重复变异系数为1.84%~8.30%,检测236份临床血清样品的阳性率为80.93%。综上,本研究E232株的成功分离,丰富了我国NDRV流行病学信息库;间接ELISA的成功建立,为临床NDRV防控提供了良好的技术手段。Novel duck reovirus(NDRV),which can infect ducklings of many breeds,leads to hemorrhagic necrosis of liver and spleen and cause immunosuppression,and has caused serious economic losses to Chinese waterfowl farming industry.However,there are still no commercial test kits on the market.In this study,a pure new duck reovirus was successfully isolated from clinical materials by plaque purification,named as E232 strain.And by usingσC protein expressed in prokaryotes as the coating antigen,an indirect ELISA was established to detect NDRV serum antibodies.The genetic evolution results show that E232 belongs to NDRV and has 96.4%-98.9%nucleotide homology with other NDRV strains.Animal regression test showed that the incidence of E232 is 100%.The spleens of the affected ducklings showed severe swelling,bleeding and necrosis,and the cloacal detoxification and pharynx detoxification reached the peak on the 5th day after infection.This assay detected NDRV antibody titer in positive sera at 1∶6400 dilution,the coefficients of variation of intra batch and inter batch repeatability tests were 1.53%-7.24%and 1.84%-8.30%.It showed 80.93%positive rate for 236 clinical serum samples but did not react with any other disease positive sera.In conclusion,the successful isolation of the E232 strain in this study has enriched the epidemiological information database of NDRV in China.The successful establishment of the indirect ELISA provides a good technical means for the prevention and control of NDRV.

关 键 词：新型鸭呼肠孤病毒 分离鉴定 σC蛋白 间接ELISA 

分 类 号：S852.659.4[农业科学—基础兽医学]