基于核苷三磷酸二磷酸水解酶的马锥虫病间接ELISA抗体检测方法的建立  

Establishment of an Indirect ELISA Antibody Detection Method for Equine Trypanosomosis Based on Nucleotide Triphosphate Diphosphohydrolase

作　　者：曾晴勤 李如松 李红英[4] 王欣乐 王莹 夏天奇 王璞 时恒枝 张路平 郑亚东 赵秀玲 宋厚辉 ZENG Qing-Qin;LI Ru-Song;LI Hong-Ying;WANG Xin-Le;WANG Ying;XIA Tian-Qi;WANG Pu;SHI Heng-Zhi;ZHANG Lu-Ping;ZHENG Ya-Dong;ZHAO Xiu-Ling;SONG Hou-Hui(Ningbo Customs Technical Center/Ningbo Key Laboratory of Port Biological and Food Safety Testing,Ningbo 315000,China;Animal Disease Prevention and Control Center of Jiangyou City,Sichuan Province,Jiangyou 621700,China;College of Animal Science and Technology&College of Veterinary Medicine/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China-Australia Joint Laboratory for Animal Health Big Data Analytics,Zhejiang A&F University,Hangzhou 311300,China;Department of Cardiology,First Hospital of Hebei Medical University,Shijiazhuang 050031,China;National Institute of Parasitic Diseases(Chinese Center for Tropical Diseases Research)/NHC Key Laboratory of Parasite and Vector Biology/WHO Collaborating Centre for Tropical Diseases/State Key Laboratory of Infectious Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Shanghai 200025,China)

机构地区：[1]宁波海关技术中心/宁波市口岸生物与食品安全检测重点实验室,宁波315000 [2]四川省江油市动物疫病预防控制中心,江油621700 [3]浙江农林大学动物科技学院&动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,杭州311300 [4]河北医科大学第一医院心脏外科,石家庄050031 [5]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心)/国家卫生健康委员会寄生虫病原与媒介生物学重点实验室/世界卫生组织热带病合作中心/传染病溯源预警与智能决策全国重点实验室,上海200025

出　　处：《农业生物技术学报》2025年第1期220-227,共8页Journal of Agricultural Biotechnology

基　　金：海关总署科研计划项目(2022HK040);宁波中盛产品检测有限公司技术开发项目(2023ZS003);上海市加强公共卫生体系建设三年行动计划(2023-2025年)重点学科项目(GWVI-11.1-12);浙江农林大学科研发展基金项目(2022LFR035)。

摘　　要：伊氏锥虫病通常称为苏拉(Surra),是由伊氏锥虫(Trypanosoma evansi)寄生于马(Equus)、骆驼(Camelus)等动物的血液而引起的一种原虫病。精准诊断是有效防控伊氏锥虫病的关键环节,但是现有诊断技术存在假阴性率高、价格昂贵等缺点。本研究在体外重组表达伊氏锥虫的核苷三磷酸二磷酸水解酶(nucleoside triphosphate diphosphohydrolase, TeNTPDase),通过优化抗原包被浓度及其条件、血清稀释度及其孵育条件、酶标二抗稀释度及其孵育条件等反应参数,建立基于TeNTPDase的马锥虫病间接ELISA抗体检测方法。结果显示,重组TeNTPDase主要以包涵体形式表达,大小约为60 kD;建立的ELISA方法在不同批次包被抗原中的变异系数为3.50%~7.32%,均小于10%,在37℃不同孵育时间内的变异系数为3.89%~14.45%,小于15%,表明所建立的方法具有较好的重复性和稳定性;利用建立的ELISA方法检测马临床血清样品,结果显示,伊氏锥虫病的阳性率为5.83%(7/120),与商品化ELISA试剂盒的阳性率(3.33%)没有显著差异,符合率为92.5%(111/120)。本研究为马锥虫病诊断和有效防控提供技术支持。Trypanosomosis(Surra)by Trypanosoma evansi,which resides in the blood,is a protozoan disease in animals such as horses(Equus)and camels(Camelus).Accurate diagnosis is a crucial step in the effective prevention and control of Surra,but the existing diagnostic techniques have many drawbacks such as high false negative rates and high prices.Based on recombinant expression of nucleoside triphosphate diphosphate hydrolase(TeNTPDase)from T.evansi,in this study,an ELISA method was established for Surra by optimizing various reaction parameters,including antigen coating concentration and conditions,serum dilution and incubation conditions,conjugated second antibody dilution and incubation conditions,and evaluated its diagnostic efficacy.The results showed that the recombinant TeNTPDase was mainly expressed in the form of inclusion bodies,with a size of approximately 60 kD.The coefficient of variation of the established ELISA method in different batches of coating antigens were 3.50%~7.32%being less than 10%,and the coefficient of variation at different incubation time points at 37℃were 3.89%~14.45%being less than 15%,indicating good repeatability and stability.Moreover,when using the established ELISA method to detect clinical serum samples of horses,it was found that the positive rate of Surra was 5.83%(7/120),slightly higher than that of the commercial test kit(3.33%),but the difference was not significant,with a coincidence rate of 92.5%(111/120).This study provides technical support for the effective prevention and control of Surra in horses.

关 键 词：伊氏锥虫病 马 核苷三磷酸二磷酸水解酶(NTPDase) ELISA 

分 类 号：S852.71[农业科学—基础兽医学]