猪丹毒杆菌抗体间接ELISA检测方法的建立
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摘要
为建立一种检测猪丹毒杆菌血清抗体的ELISA方法,本研究将猪丹毒杆菌(1a型)云南分离株ZY15074的超声裂解的全菌蛋白作为包被抗原,通过反应条件的优化,建立了猪丹毒杆菌抗体间接ELISA检测方法。特异性试验结果显示该方法与猪其它10种常见病原阳性血清均无交叉反应。该方法对阳性血清的敏感性为1/256。重复性试验结果显示批内、批间变异系数均小于10%。对160份临床血清样品比对试验结果显示该方法与国外间接ELISA方法的符合率为90.6%。本研究建立的间接ELISA方法具有较好的特异性、敏感性和重复性,可以用于猪丹毒杆菌血清抗体检测和流行病学调查。
To establish an indirect ELISA for detection serum antibody against Erysipelothrix rhusiopathiae in pigs, the indirect ELISA assay was developed using the sonicated proteins of E.rhusiopathiae strain ZY15074(type 1a) isolated from Yunnan province, and the sonicated protein was used as coated protein in this study. The results of specificity test indicated that the method was specific for detecting antibodies against E.rhusiopathiae and had no cross-reactions with other pathogeny positive serum of pigs. The results of sensitivity test indicated that the detection limit for positive serum was 1/256. The results of reproducibility test showed that the coefficient of intra-and inter-variations were less than 10%. The results of comparative test showed that the coincident rate was 90.6% of the ELISA method with the imported indirect ELISA kit for detecting 160 clinical serum samples. The results indicated that the developed indirect ELISA method could be applied in serum antibodies detection and sero-epidemiology surveillance.
To establish an indirect ELISA for detection serum antibody against Erysipelothrix rhusiopathiae in pigs, the indirect ELISA assay was developed using the sonicated proteins of E.rhusiopathiae strain ZY15074(type 1a) isolated from Yunnan province, and the sonicated protein was used as coated protein in this study. The results of specificity test indicated that the method was specific for detecting antibodies against E.rhusiopathiae and had no cross-reactions with other pathogeny positive serum of pigs. The results of sensitivity test indicated that the detection limit for positive serum was 1/256. The results of reproducibility test showed that the coefficient of intra-and inter-variations were less than 10%. The results of comparative test showed that the coincident rate was 90.6% of the ELISA method with the imported indirect ELISA kit for detecting 160 clinical serum samples. The results indicated that the developed indirect ELISA method could be applied in serum antibodies detection and sero-epidemiology surveillance.
引文
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[7]李富祥,李华春,宋建领,等.副猪嗜血杆菌的分离鉴定及16S rR NA序列分析[J].中国畜牧兽医,2012,39(8):68-72.
[8]李富祥,宋建领,李华春,等.猪源多杀性巴氏杆菌的分离鉴定及其荚膜血清型鉴定[J].上海畜牧兽医通讯,2012,(2):19-21.
[9]李富祥,赵德宏,宋建领,等.猪丹毒杆菌Taq Man荧光定量PCR检测方法的建立[J].中国预防兽医学报,2016,38(5):385-389.
[10]Tijseen P.Laboratory techniques in biochemistry and molecular biologyu:practice and theory of enzyme immunoassay[M].Amesterdam:Elsevier,1985.
[11]曹翀,曹永生,宋林,等.牛传染性鼻气管炎病gB蛋白的截短表达及间接ELISA方法的建立[J].中国预防兽医学报,2015,37(2):123-127.
[12]朱晓明,邹垚,陈攀,等.副猪嗜血杆菌不同血清型稳定表达蛋白的筛选及间接ELISA检测方法的建立[J].中国兽医科学,2015,45(01):68-74.
[13]Imada Y,Takase A,Kikuma R,et al.Serotyping of 800 strains of Erysipelothrix isolated from pigs affected with erysipelas and discrimination of attenuated live vaccine strain by genotyping[J].J Clin Microbiol,2004,42(5):2121-2126.
[14]Takahashi T,Takagi M,Sawada T,et al.Cross protection in mice and swine immunized with live erysipelas vaccine to challenge exposure with strains of Erysipelothrix rhusiopathiae of various serotypes[J].Am J Vet Res,1984,45(10):2115-2118.
[15]Imada Y,Mori Y,Daizoh M et al.Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas[J].J Clin Microbiol,2003,41(11):5015-5021.
[2]To H,Sato H,Tazumi A,et al.Characterization of Erysipelothrix rhusiopathiae strains isolated from recent swine erysipelas outbreaks in Japan[J].J Vet Med Sci,2012,74(7):949-953.
[3]Kirchhoff H,Dubenkropp H,Kerlen G,et al.Application of the indirect enzyme immunoassay for the detection of antibodies against Erysipelothrix rhusiopelathiae[J].Vet Microbiol,1985,10(6):549-559.
[4]肖国生,曹三杰,文心田.Dot PAA ELISA快速检测猪丹毒抗体方法的建立与应用[J].中国预防兽医学报,2005,27(1):59-62.
[5]姚焱彬,李倩文,杨志鹏,等.检测猪丹毒杆菌抗体重组SpaA ELISA的建立[J].微生物学通报,2017,44(3):739-748.
[6]Giménez-Lirola L G,Xiao Chao-ting,Halbur P G,et al.Development and evaluation of an enzyme-linked immunosorbent assay based on a recombinant SpaA protein(rS paA 415)for detection of anti-Erysipelothrix spp.IgG antibodies in pigs[J].J Microbiol Methods,2012,91(1):191-197.
[7]李富祥,李华春,宋建领,等.副猪嗜血杆菌的分离鉴定及16S rR NA序列分析[J].中国畜牧兽医,2012,39(8):68-72.
[8]李富祥,宋建领,李华春,等.猪源多杀性巴氏杆菌的分离鉴定及其荚膜血清型鉴定[J].上海畜牧兽医通讯,2012,(2):19-21.
[9]李富祥,赵德宏,宋建领,等.猪丹毒杆菌Taq Man荧光定量PCR检测方法的建立[J].中国预防兽医学报,2016,38(5):385-389.
[10]Tijseen P.Laboratory techniques in biochemistry and molecular biologyu:practice and theory of enzyme immunoassay[M].Amesterdam:Elsevier,1985.
[11]曹翀,曹永生,宋林,等.牛传染性鼻气管炎病gB蛋白的截短表达及间接ELISA方法的建立[J].中国预防兽医学报,2015,37(2):123-127.
[12]朱晓明,邹垚,陈攀,等.副猪嗜血杆菌不同血清型稳定表达蛋白的筛选及间接ELISA检测方法的建立[J].中国兽医科学,2015,45(01):68-74.
[13]Imada Y,Takase A,Kikuma R,et al.Serotyping of 800 strains of Erysipelothrix isolated from pigs affected with erysipelas and discrimination of attenuated live vaccine strain by genotyping[J].J Clin Microbiol,2004,42(5):2121-2126.
[14]Takahashi T,Takagi M,Sawada T,et al.Cross protection in mice and swine immunized with live erysipelas vaccine to challenge exposure with strains of Erysipelothrix rhusiopathiae of various serotypes[J].Am J Vet Res,1984,45(10):2115-2118.
[15]Imada Y,Mori Y,Daizoh M et al.Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas[J].J Clin Microbiol,2003,41(11):5015-5021.