基于溶菌酶释放蛋白的猪链球菌间接ELISA抗体检测方法的建立  

Establishment of indirect-ELISA method for detection of swine strepto coccosis based on muramidase release protein

作　　者：乔宏 龚俊 栾慧 李刚[1] 刘思国[1] 王春来[1] QIAO Hong;GONG Jun;LUAN Hui;LI Gang;LIU Si-guo;WANG Chun-lai(State Key Laboratory of Veterinary Biotechnology,Laboratory of Bacterial Diseases,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第9期912-917,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十三五”国家重点研发计划——“猪重要疫病的诊断与检测新技术研究”(2016YFD0500700)。

摘　　要：为建立猪链球菌病的快速诊断方法,本研究以纯化的原核表达的猪链球菌溶菌酶释放蛋白(MRP)为包被抗原,通过各反应条件的优化建立了检测猪链球菌(SS)血清抗体的间接ELISA方法。实验确定了最佳抗原包被浓度为0.05μg/mL、待检血清最佳稀释倍数为1:400、最佳封闭液为5%脱脂乳、兔抗猪HRP-IgG的最佳稀释倍数为1:15000、抗体阴阳性临界值为0.226。特异性试验结果显示,利用本研究建立的ELISA方法检测100份背景清晰的阴性血清,检出其中97份阴性血清,3份阳性血清,特异性为97%。利用该ELISA方法检测副猪嗜血杆菌、胸膜肺炎放线杆菌、多杀巴氏杆菌、大肠杆菌、猪传染性胃肠炎病毒、轮状病毒、流行性腹泻病毒、猪繁殖与呼吸综合征病毒阳性血清,结果显示均为阴性,而猪链球菌2型(SS2)、SS7、SS9以及SS12阳性血清的检测结果均为阳性,表明该方法具有较强的特异性。敏感性试验结果显示,利用本研究建立的ELISA方法检测50份背景明确的阳性血清,检出其中48份阳性血清,2份阴性血清,敏感性为96%。当SS2阳性血清稀释倍数达1:3200时仍为阳性结果,表明该方法具有较高的敏感。重复性试验结果显示,该ELISA方法批内、批间最大变异系数分别为4.70%和7.20%,表明该方法的重复性较好。对100份临床猪血清样品检测结果显示,本研究建立的ELISA方法与玻片凝集试验的符合率为85.3%,符合率较高。对440份来自东北三省部分地区猪场的血清样品检测结果显示,SS抗体阳性率为27.7%(122/440)。表明,东北三省部分地区猪场SS的感染率较高。上述结果表明,本研究建立的间接ELISA方法可以用于SS2、SS7、SS9以及SS12血清抗体的检测,为SS病的血清流行病学调查提供了可靠的技术手段。To establish a rapid method for the diagnosis of swine streptococcosis,the prokaryotically expressed muramidase releasing protein(MRP)of streptococcus suis was purified and used as the coating antigen to establish an indirect ELISA method for the detection of sera against streptococcus suis.There action conditions were determined after optimization,the optimal antigen coating concentration was 0.05μg/mL,the optimal dilution factor of the serum for testing was 1:400,the optimal blocking solution was 5%skim milk,and the optimal dilution factor of rabbit anti-swine HRP-IgG was 1:15000,the positive cut-off threshold was 0.226.Specific test results showed that the ELISA method established in this study was used to detect 100 negative sera with a clear back ground,among which 97 negative sera and 3 positive sera were identified,with a specificity of 97%.The ELISA method was employed to detect Haemophilus parasuis(HPS),Actinobacillus pleuropneumoniae(APP),Pasteurella multocida(PM),E.coli,Porcine infectious gastroenteritis virus(TGEV),Rotavirus(RV),porcine epidemic diarrhea virus(PEDV),porcine blue ear virus(PRRSV)positive sera,the results were all negative,and streptococcus suis type 2(SS2),type 7(SS7),type 9(SS9)and type 12(SS12)positive sera were all positive,indicating that the method has high specificity.The sensitivity test results showed that the established ELISA method was used to detect 50 positive sera with a clear background,among which 48 positive and 2 negative sera were detected,with a sensitivity of 96%.The result remained positive even the SS2 positive serum dilution ratio reached 1:3200,indicating that the method has higher sensitivity.The repeatability test showed that the maximum coefficients of variation of the intra-and inter-assay were 4.70%and 7.20%,indicating that the method has good repeatability.The coincidence rate between the ELISA method and glassagglutination test was 85.3%,when 100 clinical pig serum samples were tested.Serum samples from 440 clinical pig were tested by the ELISA method,a

关 键 词：猪链球菌 重组MRP蛋白 间接ELISA 

分 类 号：S852.61[农业科学—基础兽医学]