猪化脓隐秘杆菌抗体间接ELISA检测方法的建立
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摘要
为建立检测化脓隐秘杆菌(T.pyogenes)血清抗体的间接ELISA方法,本研究以超声破碎处理T.pyogenes分离株TP-2849 (NZ_CP029004)的全菌蛋白作为包被抗原,经反应条件优化建立了T.pyogenes抗体间接ELISA检测方法。该方法与7种常见病原阳性血清均无交叉反应,特异性较强;敏感性试验显示阳性血清的检测下限为1∶128,敏感性较高;组内、组间变异系数均小于9.5%,重复性较好。对吉林省某养殖场100份猪血清检测结果显示,该方法与PCR检测方法总符合率为94%。本研究建立的间接ELISA方法为T.pyogenes抗体检测提供了一种新手段。
To establish an indirect ELISA(iELISA) for detecting antibodies against Trueperella pyogenes, we used the sonicated lysate of T.pyogenes TP-2849 strain(NZ_CP029004) as the coating antigen and optimized reaction conditions. The specificity test result showed that the iELISA had no cross-reactions with the other seven common pathogenic positive sera of H.parasuis, E.coli, P.multocida, CSFV, PRRSV, PPV and PRV, which showed the established assay was high specificity for detection of the antibody against T.pyogenes. The detection limit of the iELISA for positive serum was 1 ∶128. The coefficient of intra-variations and inter-variations were both less than 9.5%. Above all, the established iELISA provide a new method for detection of the antibody against T.pyogenes.
To establish an indirect ELISA(iELISA) for detecting antibodies against Trueperella pyogenes, we used the sonicated lysate of T.pyogenes TP-2849 strain(NZ_CP029004) as the coating antigen and optimized reaction conditions. The specificity test result showed that the iELISA had no cross-reactions with the other seven common pathogenic positive sera of H.parasuis, E.coli, P.multocida, CSFV, PRRSV, PPV and PRV, which showed the established assay was high specificity for detection of the antibody against T.pyogenes. The detection limit of the iELISA for positive serum was 1 ∶128. The coefficient of intra-variations and inter-variations were both less than 9.5%. Above all, the established iELISA provide a new method for detection of the antibody against T.pyogenes.
引文
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[2]Miranda J,Bakheit M A,Liu Zhi-shui,et al.Development of a recombinant indirect ELISA for the diagnosis of Theileria sp.(China)infection in small ruminants[J].Parasutol Res,2006,98(6):561-567.
[3]马福利,王丽荣,董永军,等.副猪嗜血杆菌抗体间接ELISA检测方法的建立及应用[J].西北农业学报,2015,24(5):29-33.
[4]曹俊,卢锦标,谢安平,等.耻垢分枝杆菌与结核分枝杆菌抗原的交叉免疫应答研究[J].中华微生物学和免疫学杂志,2017,37(4):275-280.
[5]李娇阳,董虎,郭慧琛,等.伪狂犬病病毒gB抗原的可溶性原核表达及纯化条件的优化[J].中国兽医科学,2018,(2):175-181.
[6]李富祥,朱凤琼,王艳芬,等.猪丹毒杆菌抗体间接ELISA检测方法的建立[J].中国预防兽医学报,2018,(4):311-315.
[7]李雪秋.小鼠血清中弓形虫Peroxiredoxin抗原间接ELISA检测方法的建立及其血清流行病学应用[D].太原:山西医科大学,2010.
[8]Duarte V D S,Dias R S,Kropinski A M,et al.A T4virus,prevents biofilm formation by Trueperella pyogenes[J].Vet Microbiol,2018,218:45-51.
[9]Gouletsou P G,Fthenakis G C,Cripps P J,et al.Experimentally induced orchitis associated with Arcanobacterium pyogenes:Clinical,ultrasonographic,seminological and pathological features[J].Theriogenology,2004,62(7):1307-1328.
[10]姚焱彬.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.