气肿疽梭菌多克隆抗体的制备及Dot-ELISA检测方法的建立
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摘要
为建立快速有效的气肿疽检测方法,制备气肿疽梭菌多克隆抗体。以斑点酶联免疫吸附方法,筛选出最佳工作条件,确定抗体最佳稀释度为1∶1 024;可检测到最低菌体抗原量为3.125μg,最低分泌抗原量为1.89μg;最适酶标二抗浓度为1∶4 000。应用该方法检测20份死于气肿疽的豚鼠脏器,其中肝脏、脾脏、心脏、肺脏、肾脏、肌肉阳性率分别为95%、95%、90%、90%、85%和90%;与琼脂免疫扩散试验(AGID)法相比,灵敏度是其40倍。该试验成功建立了检测气肿疽病原体的Dot-ELISA方法。
In order to establish a rapid and effective method for the detection of emphysema,and to prepare polyclonal antibody for pneumonitis,our study developed a Dot-ELISA detecting method and selected the optimum working conditions.The optimal dilution ratio of the antibody was 1∶1,024.The minimum amount of antigen was 3.125μg and the lowest secretion antigen was 1.89μg.The optimal concentration of enzyme was 1∶4 000.Twenty guinea pigs samples were detected using this Dot-ELISA method,and the positive rate of liver,spleen,heart,lung,kidney and muscle were 95%,95%,90%,90%,85% and90%.The sensitivity of this method is 40 times than AGID.The results show that the Dot-ELISA method was established successfully to detect the pathogen.
In order to establish a rapid and effective method for the detection of emphysema,and to prepare polyclonal antibody for pneumonitis,our study developed a Dot-ELISA detecting method and selected the optimum working conditions.The optimal dilution ratio of the antibody was 1∶1,024.The minimum amount of antigen was 3.125μg and the lowest secretion antigen was 1.89μg.The optimal concentration of enzyme was 1∶4 000.Twenty guinea pigs samples were detected using this Dot-ELISA method,and the positive rate of liver,spleen,heart,lung,kidney and muscle were 95%,95%,90%,90%,85% and90%.The sensitivity of this method is 40 times than AGID.The results show that the Dot-ELISA method was established successfully to detect the pathogen.
引文
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[11]张皓,任春宇,车达,等.气肿疽检测方法概述[J].动物医学进展,2017,38(9):119-121.
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[13]Mattar M A,Cortinas T I,Stefanini A M.Extracellular proteins of Clostridium chauvoei are protective in a mouse model[J].Acta Vet Hung,2007,(55):159-170.
[14]Vilei E M,Johansson A,Schlatter Y,et al.Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei[J].Vet Res,2011,(42):2.
[15]Sathish S,Swaminathan K.Molecular characterization of the diversity of Clostridium chauvoei isolatescollected from two bovine slaughterhouses:Analysis of cross-contamination[J].Anaerobe,2008,(14):190-199.
[16]彭小兵,李旭妮,王楠,等.二重PCR方法鉴别气肿疽梭菌和腐败梭菌[J].中国兽药杂志,2011,45(3):20-22.
[17]姜丹丹,金鑫,陈莹莹,等.气肿疽梭菌套式PCR检测方法的建立[J].中国兽医科学,2010,40(11):1171-1174.
[18]李云霄.Dot-ELISA检测牛D型产气荚膜梭菌病抗体的研究[D].延吉:延边大学,2008.
[19]Sasaki Y,Yamamoto K,Amimoto K,et al.Amplification of the 16S-23SrDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum[J].Res Vet Sci,2001,71(3):227-229.
[20]Sasaki Y,KojimaA,Aoki H,et al.Phylogenetic analysis and PCR detection of Clostridium chauvoei,Clostridium haemolyticum,Clostridium novyi types A and B,and Clostridium septicum based on the flagellin gene[J].Vet Microbiol,2002,86(3):257-267.
[2]张守发,张国宏.猪附红细胞体Dot-ELISA检测方法的建立[J].中国预防兽医学报,2006(01):96-98.
[3]Russo V C,Gluckman P D,Feldman E L,et al.The insulin-like growth factor system and its pleiotropic functions in brain[J].Endocrine Reviews,2005,26(7):916-943.
[4]Groseth P K,Ersdal C,Bjelland A M,et al.Large outbreak of blackleg in housed cattle[J].Vet Rec,2011,169(13):339.
[5]周金玲,吴丹丹,周玉龙,等.气肿疽梭菌HLJ-1株分离鉴定及系统进化分析[J].黑龙江八一农垦大学学报,2016,28(06):83-88.
[6]车达,金鑫,陈莹莹,等.延边黄牛气肿疽间接ELISA诊断方法的建立[J].安徽农业科学,2011,39(02):1042-1044.
[7]Lange M,Neubauer H,Seyboldt C.Development and validation of a multiplex real-time PCR for detection of Clostridium chauvoei and Clostridium septicum[J].Mol Cell Probes,2010(24):204-210.
[8]张永佳,张皓,李成辉,等.延边株气肿疽梭菌鞭毛蛋白FLiA基因克隆及生物信息学分析[J].延边大学农学学报,2017,39(2):35–40.
[9]王兴红.羊气肿疽的流行特点、临床表现、实验室诊断及防控[J].现代畜牧科技,2017,33(9):119.
[10]云巾宴,任春宇,金鑫,等.气肿疽梭菌PCR检测方法的建立[J].江苏农业科学,2015,43(10):53-55.
[11]张皓,任春宇,车达,等.气肿疽检测方法概述[J].动物医学进展,2017,38(9):119-121.
[12]Mattar M A,Cortinas T I,deGuzmanA M.Immunogenic protein variations of Clostridium chauvoei cellular antigens associated with the culture growth phase[J].FEMS Immunol Med Microbiol,2002,33(1):9-14.
[13]Mattar M A,Cortinas T I,Stefanini A M.Extracellular proteins of Clostridium chauvoei are protective in a mouse model[J].Acta Vet Hung,2007,(55):159-170.
[14]Vilei E M,Johansson A,Schlatter Y,et al.Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei[J].Vet Res,2011,(42):2.
[15]Sathish S,Swaminathan K.Molecular characterization of the diversity of Clostridium chauvoei isolatescollected from two bovine slaughterhouses:Analysis of cross-contamination[J].Anaerobe,2008,(14):190-199.
[16]彭小兵,李旭妮,王楠,等.二重PCR方法鉴别气肿疽梭菌和腐败梭菌[J].中国兽药杂志,2011,45(3):20-22.
[17]姜丹丹,金鑫,陈莹莹,等.气肿疽梭菌套式PCR检测方法的建立[J].中国兽医科学,2010,40(11):1171-1174.
[18]李云霄.Dot-ELISA检测牛D型产气荚膜梭菌病抗体的研究[D].延吉:延边大学,2008.
[19]Sasaki Y,Yamamoto K,Amimoto K,et al.Amplification of the 16S-23SrDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum[J].Res Vet Sci,2001,71(3):227-229.
[20]Sasaki Y,KojimaA,Aoki H,et al.Phylogenetic analysis and PCR detection of Clostridium chauvoei,Clostridium haemolyticum,Clostridium novyi types A and B,and Clostridium septicum based on the flagellin gene[J].Vet Microbiol,2002,86(3):257-267.