猪圆环病毒2型抗原乳胶免疫层析试纸的构建与应用  

Development and application of latex immunitychromatography test strip for porcine circovirus type 2 antigen

作　　者：陶思锐 查银河 何玉龙 舒建洪[1,3] TAO Si-rui;ZHA Yin-he;HE Yu-long;SHU Jian-hong(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China;Zhejiang Sci-Tech University Shaoxing Academy of Biomedicine,Shaoxing 312000,China;Zhejiang HomSun Biotechnology INC.,LTD.,Shaoxing 312000,China)

机构地区：[1]浙江理工大学生命科学与医药学院,浙江杭州310018 [2]浙江理工大学绍兴生物医药研究院,浙江绍兴312000 [3]浙江洪晟生物科技股份有限公司,浙江绍兴312000

出　　处：《中国兽医科学》2022年第8期999-1007,共9页Chinese Veterinary Science

基　　金：绍兴市科技计划项目(2020B33002);浙江省重点研发计划项目(2019C02043)。

摘　　要：为构建一种强特异性、高灵敏度、检测快速、操作简便的猪圆环病毒2型(PCV2)抗原诊断方法,本研究通过原核表达系统表达Cap蛋白并制备相应的多克隆抗体。用多克隆抗体和乳胶微球制备免疫探针,将单克隆抗体和羊抗兔二抗分别包被于硝酸纤维膜(NC膜)上的检测线(T线)和质控线(C线)。对工艺参数进行优化后进行试纸条的性能验证,结果显示,该试纸仅在PCV2抗原阳性血清下呈现阳性结果,与其他病毒抗原阳性血清无交叉反应;检出限为0.5 ng/m L;对150份临床样本进行检测,以PCR检测试剂盒的结果为金标准,试纸条的样本检出率为93.33%(140/150);该试纸在常温、20%湿度环境下可保存至少12个月。结果表明,本研究构建的PCV2抗原检测试纸具有强特异性、高灵敏度、良好稳定性、检测快速、操作简便的特点,可用于猪圆环病毒2型感染的早期诊断,对猪圆环病毒2型的预防具有重要意义。The purpose of this study is to build a simple,fast,specific and sensitive method which can detect the antigen against porcine circovirus type 2(PCV2).Recombinant Cap protein was expressed and purified by prokaryotic expression system,and rabbits were immunized to obtain the polyclonal antibody against recombinant Cap protein.The polyclonal antibody and latex microspheres were used to prepare immunoprobe.Furthermore,the monoclonal antibody and goat anti rabbit Ig G were coated on the nitrocellulose membrane as the test line and control line.The conditions of manufacturing process were optimized.After performance validation experiments,positive results can be showed when PCV2 positive serums were tested.There was no crossing-reaction with other positive serum,the developed test paper had high specificity.The limit of detection(LOD) of test paper was 0.5 ng/m L.150 clinical specimens were detected by developed test paper and PCR method,when PCR results werer using as gold standard,the detection rate of PCV2 was 93.33%(140/150) by immunitychromatography test strip.The test papers could be stored for 12 months at room temperature.The above results showed that the test paper constructed in this study had high specificity and sensitivity and were easy to operate good stability.Therefore,the developed test papers could be used to diagnose PCV2 at early diagnosis.The developed test paper will be of great significance for monitoring and preventing against PCV2.

关 键 词：猪圆环病毒2型 原核表达 乳胶微球 免疫层析 

分 类 号：S852.659.2[农业科学—基础兽医学]