猪旋毛虫抗体管式化学发光免疫分析检测方法的建立与应用  

Establishment and Application of a Tube-Based Chemiluminescence Immunoassay Method for Detecting Antibodies Against Trichinella spiralis in Pigs

作　　者：钱艳红 宋帅 温肖会 牛瑞辉 杨燕秋 郑博彬 袁子国[2] 罗胜军 QIAN YanHong;SONG Shuai;WEN XiaoHui;NIU RuiHui;YANG YanQiu;ZHENG BoBin;YUAN ZiGuo;LUO ShengJun(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Guangdong Province Key Laboratory of Livestock Diseases Prevention/Guangdong Scientific Observation and Experiment Station for Veterinary Drugs and Diagnostic Technologies,Ministry of Agriculture and Rural Areas,Guangzhou 510640;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广州510640 [2]华南农业大学兽医学院,广州510642

出　　处：《中国农业科学》2024年第22期4578-4588,共11页Scientia Agricultura Sinica

基　　金：猪禽种业全国重点实验室开放课题(2023QZ-NK14);广东省科技计划项目(2021B1212050021)。

摘　　要：【背景】旋毛虫(Trichinella spiralis)是一种食源性人兽共患寄生线虫,被列为世界七大食源性寄生虫,在家畜中,旋毛虫主要感染猪,并且能够通过猪肉及副产品传播给人类,不仅给我国生猪养殖产业带来了巨大的经济损失,而且严重危害人类健康和公共卫生安全。【目的】建立一种灵敏度高,特异性强,快速自动化的猪旋毛虫血清学检测方法,为阻止人兽共患寄生虫病的传播提供帮助。【方法】利用基因克隆和原核表达技术构建原核表达载体,通过诱导表达获得旋毛虫14-3-3重组蛋白,并验证其反应原性。用制备的重组蛋白包被磁性微粒,以此为检测抗原建立化学发光免疫分析方法,并对建立的方法进行条件优化。最后对优化后的方法进行临界值的界定及特异性、敏感性和重复性的确定;通过对来自广东省和贵州省不同养猪场的1000份猪临床血清样本进行检测,评估该检测方法的实用性及符合率。【结果】成功表达并纯化出Ts14-3-3重组蛋白,该蛋白能特异性识别猪旋毛虫阳性血清中的抗体。建立的检测方法经过优化,结合缓冲液最佳pH为5,Ts14-3-3重组蛋白最佳包被量为5μg·mg^(-1)-beads,活化剂最佳使用量为50μg·mg^(-1)-beads,1%BSA封闭效果最好,酶标二抗最佳稀释度为1﹕40000,最佳反应时间为10 min,待检样本最佳孵育时间为5 min,最佳酶促反应时间为2 min。最后经过评估,该检测方法的临界化学发光值为374185 RLU,与猪弓形虫、猪球虫、猪结肠小袋纤毛虫、猪繁殖与呼吸综合征、经典猪瘟、猪伪狂犬和猪口蹄疫抗体均无交叉反应,该检测方法与现有ELISA检测方法相比敏感性更高,批内和批间重复性均小于10%,检测的1000份临床猪血清样本中旋毛虫抗体阳性率为0.6%,与市售ELISA试剂盒检出总符合率为98.18%。【结论】成功建立了猪旋毛虫抗体管式化学发光免疫分析检测方法,该检测方法特异【Background】Trichinella spiralis is a foodborne zoonotic parasitic nematode,listed as one of the seven major foodborne parasites in the world.In domestic animals,Trichinella spiralis mainly infects pigs and can be transmitted to humans through pork and by-products.It not only causes huge economic losses to China's pig farming industry,but also seriously endangers human health and public health safety.【Objective】This study aimed to establish a highly sensitive,specific,and rapid automated serological detection method for Trichinella spiralis in pigs.【Method】This study utilized gene cloning and prokaryotic expression techniques to construct a prokaryotic expression vector,obtained the recombinant protein of Trichinella spiralis 14-3-3 through induction of expression,and verified its reactivity.A chemiluminescence enzyme immunoassay method was established by encapsulating magnetic particles with prepared recombinant proteins as detection antigens,and the conditions for the established method were optimized.Finally,based on the optimized method,the critical values were defined and the specificity,sensitivity,and repeatability were determined;by testing 1000 clinical serum samples of pigs from different pig farms in Guangdong and Guizhou provinces,the practicality and compliance rate of this testing method were evaluated.【Result】The recombinant protein Ts14-3-3 was successfully expressed and purified,which could specifically recognize antibodies in positive serum of Trichinella spiralis in pigs.The established detection method has been optimized,with the optimal pH value of the buffer solution being 5 and the optimal encapsulation amount of the Ts14-3-3 recombinant protein being 5μg·mg^(-1)-beads,the optimal dosage of the activator was 50μg·mg^(-1)-beads,and 1%BSA has the best sealing effect.The optimal dilution for enzyme-labeled antibodies was 1:40000,the optimal reaction time was 10 minutes,the optimal incubation time for the sample to be tested was 5 minutes,and the optimal enzymatic reaction t

关 键 词：旋毛虫 血清学诊断 Ts14-3-3 原核表达 管式化学发光免疫分析 

分 类 号：S858.28[农业科学—临床兽医学]