Ⅰ群禽腺病毒4型IgG抗体间接ELISA检测方法的建立
|
摘要
利用纯化的Ⅰ群禽腺病毒4型重组Hexon蛋白,通过各反应条件筛选,建立了Ⅰ群禽腺病毒4型IgG抗体间接ELISA检测方法。结果显示,抗原最佳包被浓度为1μg/mL;最佳封闭液为1%牛血清白蛋白;待检血清最佳作用浓度是400倍稀释,37℃孵育60 min;酶标抗体的最佳作用条件为稀释度1∶8000,37℃孵育60 min;最佳显色时间为10 min。用所建方法对160只临床鸡只进行检测,同时与拭子PCR检测对比,一致性较好,表明该方法可有效用于临床检测。
With various reaction conditions being screened,an indirect ELISA was established for detection of group Ⅰ Fowl adenovirus serotype 4 IgG antibody by using the purified recombination Hexon protein. The results showed that the optimal concentration of the coating antigen was 1 μg/mL; the best blocking solution was 1% bovine serum albumin; the optimal concentration of the serum to be tested was 400-fold dilution,and the optimal incubation time of the serum was 60 minutes at 37 ℃; the optimal conditions of the enzyme-labeled antibody were 1 ∶8000 dilution,and incubated for 60 minutes at 37 ℃; the optimal chromogenic time was 10 minutes. The established method was used to detect 160 clinical chickens and compared with the swab PCR test,the results were consistent,indicating that the proposed method can be used for clinical detection effectively.
With various reaction conditions being screened,an indirect ELISA was established for detection of group Ⅰ Fowl adenovirus serotype 4 IgG antibody by using the purified recombination Hexon protein. The results showed that the optimal concentration of the coating antigen was 1 μg/mL; the best blocking solution was 1% bovine serum albumin; the optimal concentration of the serum to be tested was 400-fold dilution,and the optimal incubation time of the serum was 60 minutes at 37 ℃; the optimal conditions of the enzyme-labeled antibody were 1 ∶8000 dilution,and incubated for 60 minutes at 37 ℃; the optimal chromogenic time was 10 minutes. The established method was used to detect 160 clinical chickens and compared with the swab PCR test,the results were consistent,indicating that the proposed method can be used for clinical detection effectively.
引文
[1]袁万哲，李玉保，王建昌，等．鸡心包积液-肝炎综合征的初步研究[J]．中国兽医科学，2016(2):157-160.Yuan W Z,Li Y B,Wang J C,et al.Preliminary study on chicken pericardial effusion-hepatitis syndrome[J].Chinese Journal of Veterinary Science,2016(2):157-160．
[2]Griffin B D,Nagy E.Coding potential and transcript analysis of fowl adenovirus 4:insight into upstream ORFs as common sequence features in adenoviral transcripts[J].Journal of General Virology,2011,92(Pt 6):1260-1272．
[3]Niczyporuk J S.Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland[J].Archives of Virology,2016,161(1):33-42．
[4]Mittal D,Jindal N,Tiwari A K,et al.Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens[J].Virus Disease,2014,25(1):114-119．
[5]Li H,Wang J,Qiu L,et al.Fowl adenovirus species C serotype4 is attributed to the emergence of hepatitis-hydropericardium syndrome in chickens in China.[J].Infection,Genetics and Evolution:Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases,2016,45:230-241．
[6]刁有祥．I群禽腺病毒感染的流行情况与防控[J]．北方牧业，2016(10):21-21.Diao Y X.Epidemic situation and prevention and control of group I avian adenovirus infection[J].Northern Animal Husbandry,2016(10):21-21．
[7]牛登云，沈元，王蕊，等．2015年我国Ⅰ群禽腺病毒分子流行病学调查[J]．中国家禽，2016,38(9):65-68.Niu D Y,Shen Y,Wang R,et al.Molecular epidemiological survey of group I avian adenovirus in China in 2015[J].China Poultry,2016,38(9):65-68．
[8]Chandr R,Gomez-Villamandos J C.Ultrastructural changes in the liver of birds experimentally infected with the agent of hydropericariium syndrome[J].Acta Virologica,2001,45(2):129．
[9]Horst L,Christopher R,Roland W,et al.Hydropericardium syndrome:current state and future developments[J].Archives of Virology,2013,158(5):921-931．
[10]Cai F,Weber J M.Organization of the avian adenovirus genome and the structure of its endopeptidase[J].Virology,1993,196(1):358-362．
[11]文艳玲，谢芝勋，黄琦，等．禽腺病毒1型和4型六邻体蛋白抗原表位和密码子偏爱性分析[J]．动物医学进展，2007,28(11):17-20.Wen Y L,Xie Z X,Huang Q,et al.Epitope and codon preference analysis of avian adenovirus type 1 and type 4 Hexon proteins[J].Progress in Animal Medicine,2007,28(11):17-20．
[12]刘延珂，万文妍，王贝贝，等．禽腺病毒4型抗体间接ELISA检测方法的建立[J]．中国预防兽医学报，2017(11):902-906.Liu Y K,Wan W Y,Wang B B,et al.Establishment of an indirect ELISA method for detection of avian adenovirus type 4antibody[J].Chinese Journal of Preventive Veterinary Medicine,2017(11):902-906．
[13]国纪垒，刁有祥．I群禽腺病毒山东株的分离鉴定及Hexon基因的克隆与分析[C]∥中国畜牧兽医学会2011学术年会，2011:1773-1777.Guo J L,Diao Y X.Isolation and identification of avian adenovirus I strain from Shandong province and cloning and analysis of Hexon gene[C]∥2011 Annual Meeting of Chinese Society of Animal Husbandry and Veterinary Science,2011:1773-1777．
[14]吴烨挺．禽腺病毒4型的分离鉴定及毒种免疫原性研究[D]．内蒙古农业大学，2017.Wu Y T.Isolation and identification of avian adenovirus type 4 and study on its immunogenicity[D].Inner Mongolia Agricultural University,2017．
[15]Ye J,Liang G,Zhang J,et al.Outbreaks of serotype 4 fowl adenovirus with novel genotype,China[J].Emerging Microbes&Infections,2016,5(5):e50．
[16]Zhao J,Zhong Q,Zhao Y,et al.Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China[J].Plos One,2015,10(7):e0133073．
[17]汪凯，蔡骁垚，叶建强，等．新型鸡心包炎-包涵体肝炎病毒的分离及鉴定[J]．中国动物传染病学报，2016,24(4):1-6.Wang K,Cai X J,Ye J Q,et al.Isolation and identification of novel chicken pericarditis--inclusion body hepatitis virus[J].Chinese Journal of Zoonotic Diseases,2016,24(4):1-6．
[18]赵静，冯金玲，靳继惠，等．2016年鸡重要疫病流行动态分析[J]．中国家禽，2016,38(12):69-72.Zhao J,Feng J L,Jin J H,et al.Dynamics analysis of important chicken epidemic in 2016[J].China Poultry,2016,38(12):69-72．
[19]吴爱华．禽腺病毒流行与危害[J]．中国畜禽种业，2018,14(9):160-161.Wu A H.Prevalence and harm of avian adenovirus[J].China Livestock and Poultry Seed Industry,2018,14(9):160-161．
[20]Teng Z,Jin Q,Ding P,et al.Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China[J].Virology Journal,2016,13(1):188．
[21]Yu B,Dong J,Wang C,et al.Characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera[J].Virology,2013,437(2):118-123.
[2]Griffin B D,Nagy E.Coding potential and transcript analysis of fowl adenovirus 4:insight into upstream ORFs as common sequence features in adenoviral transcripts[J].Journal of General Virology,2011,92(Pt 6):1260-1272．
[3]Niczyporuk J S.Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland[J].Archives of Virology,2016,161(1):33-42．
[4]Mittal D,Jindal N,Tiwari A K,et al.Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens[J].Virus Disease,2014,25(1):114-119．
[5]Li H,Wang J,Qiu L,et al.Fowl adenovirus species C serotype4 is attributed to the emergence of hepatitis-hydropericardium syndrome in chickens in China.[J].Infection,Genetics and Evolution:Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases,2016,45:230-241．
[6]刁有祥．I群禽腺病毒感染的流行情况与防控[J]．北方牧业，2016(10):21-21.Diao Y X.Epidemic situation and prevention and control of group I avian adenovirus infection[J].Northern Animal Husbandry,2016(10):21-21．
[7]牛登云，沈元，王蕊，等．2015年我国Ⅰ群禽腺病毒分子流行病学调查[J]．中国家禽，2016,38(9):65-68.Niu D Y,Shen Y,Wang R,et al.Molecular epidemiological survey of group I avian adenovirus in China in 2015[J].China Poultry,2016,38(9):65-68．
[8]Chandr R,Gomez-Villamandos J C.Ultrastructural changes in the liver of birds experimentally infected with the agent of hydropericariium syndrome[J].Acta Virologica,2001,45(2):129．
[9]Horst L,Christopher R,Roland W,et al.Hydropericardium syndrome:current state and future developments[J].Archives of Virology,2013,158(5):921-931．
[10]Cai F,Weber J M.Organization of the avian adenovirus genome and the structure of its endopeptidase[J].Virology,1993,196(1):358-362．
[11]文艳玲，谢芝勋，黄琦，等．禽腺病毒1型和4型六邻体蛋白抗原表位和密码子偏爱性分析[J]．动物医学进展，2007,28(11):17-20.Wen Y L,Xie Z X,Huang Q,et al.Epitope and codon preference analysis of avian adenovirus type 1 and type 4 Hexon proteins[J].Progress in Animal Medicine,2007,28(11):17-20．
[12]刘延珂，万文妍，王贝贝，等．禽腺病毒4型抗体间接ELISA检测方法的建立[J]．中国预防兽医学报，2017(11):902-906.Liu Y K,Wan W Y,Wang B B,et al.Establishment of an indirect ELISA method for detection of avian adenovirus type 4antibody[J].Chinese Journal of Preventive Veterinary Medicine,2017(11):902-906．
[13]国纪垒，刁有祥．I群禽腺病毒山东株的分离鉴定及Hexon基因的克隆与分析[C]∥中国畜牧兽医学会2011学术年会，2011:1773-1777.Guo J L,Diao Y X.Isolation and identification of avian adenovirus I strain from Shandong province and cloning and analysis of Hexon gene[C]∥2011 Annual Meeting of Chinese Society of Animal Husbandry and Veterinary Science,2011:1773-1777．
[14]吴烨挺．禽腺病毒4型的分离鉴定及毒种免疫原性研究[D]．内蒙古农业大学，2017.Wu Y T.Isolation and identification of avian adenovirus type 4 and study on its immunogenicity[D].Inner Mongolia Agricultural University,2017．
[15]Ye J,Liang G,Zhang J,et al.Outbreaks of serotype 4 fowl adenovirus with novel genotype,China[J].Emerging Microbes&Infections,2016,5(5):e50．
[16]Zhao J,Zhong Q,Zhao Y,et al.Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China[J].Plos One,2015,10(7):e0133073．
[17]汪凯，蔡骁垚，叶建强，等．新型鸡心包炎-包涵体肝炎病毒的分离及鉴定[J]．中国动物传染病学报，2016,24(4):1-6.Wang K,Cai X J,Ye J Q,et al.Isolation and identification of novel chicken pericarditis--inclusion body hepatitis virus[J].Chinese Journal of Zoonotic Diseases,2016,24(4):1-6．
[18]赵静，冯金玲，靳继惠，等．2016年鸡重要疫病流行动态分析[J]．中国家禽，2016,38(12):69-72.Zhao J,Feng J L,Jin J H,et al.Dynamics analysis of important chicken epidemic in 2016[J].China Poultry,2016,38(12):69-72．
[19]吴爱华．禽腺病毒流行与危害[J]．中国畜禽种业，2018,14(9):160-161.Wu A H.Prevalence and harm of avian adenovirus[J].China Livestock and Poultry Seed Industry,2018,14(9):160-161．
[20]Teng Z,Jin Q,Ding P,et al.Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China[J].Virology Journal,2016,13(1):188．
[21]Yu B,Dong J,Wang C,et al.Characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera[J].Virology,2013,437(2):118-123.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |