牛病毒性腹泻病毒抗原胶体金快速检测试纸条的研制  

Development of colloidal gold test strip for rapid detection of BVDV antigen

作　　者：卜庆龙 左之才[1] 才冬杰 叶刚 田斌 李伟强 苟丽萍[1] 王娅[1] 邓俊良[1] BU Qing-long;ZUO Zhi-cai;CAI Dong-jie;YE Gang;TIAN Bin;LI Wei-qiang;GOU Li-ping;WANG Ya;DENG Jun-liang(School of Veterinary Medicine,Sichuan Agricultural University/Sichuan Key Laboratory of Animal Disease and Human Health,Chengdu 611130,China;Shandong Animal Husbandry and Veterinary Vocational College,Weifang 261061,China)

机构地区：[1]四川农业大学动物医学院/动物疫病与人类健康四川省重点实验室,四川成都611130 [2]山东畜牧兽医职业学院宠物科技学院,山东潍坊261061

出　　处：《中国预防兽医学报》2023年第12期1253-1257,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划资助(2021YFD1600200);国家现代农业产业技术体系资助(肉牛牦牛,CARS-37);四川省自然科学基金(2022NSFSC1620)。

摘　　要：为建立一种快速准确且便携的检测牛病毒性腹泻病毒(BVDV)的方法,本研究将制备的BVDV单克隆抗体(MAb)作为检测线(T线),以纯化的免源多克隆抗体作为金标蛋白,以羊抗兔IgG-HRP作为质控线(C线),组装成双抗体夹心胶体金层析试纸条,并优化各反应条件。结果显示,最适金标蛋白标记量为30μg/mL,BVDV MAb和羊抗兔IgG-HRP最佳浓度均为0.5 mg/mL。利用猪瘟病毒(CSFV)、牛传染性鼻气管炎病毒(IBRV)、牛丘疹性口炎病毒(BPSV)及BVDV临床分离株评估该试纸条的特异性,结果显示,除阳性对照和BVDV临床分离株检测为阳性外,其余病毒均为阴性,表明该试纸条的特异性较强,无交叉反应。将BVDV-NADL株(10^(6.6)ELD_(50)/mL)10倍倍比稀释后,利用该试纸条检测,结果显示其最低检出量为10^(4.6)ELD_(50)/mL。利用同一批次和不同批次制备的试纸条对CSFV、IBRV、BPSV、BVDV临床分离株检测,结果显示检测结果均一致,表明该试纸条的重复性良好。将制备的试纸条应用于检测临床采集的68份牛血清样品,并以RT-PCR方法同时检测,结果显示胶体金试纸条对BVDV的阳性检出率为17.5%(12/68),RT-PCR对BVDV的阳性检出率为26.5%(18/68),两者之间的总符合率为88.24%。本研究于国内首次采用双抗体夹心方式制备了检测BVDV的胶体金免疫层析试纸条,该方法方便快捷,特异性及敏感性均较好,适宜于牛场BVDV的快速筛选,为进一步研制BVDV胶体金检测试剂盒提供了实验依据。In order to establish a clinical rapid and portable method for the detection of bovine viral diarrhea virus(BVDV),a monoclonal antibody(MAb)against BVDV prepared in our laboratory was used as the T line,the gold-labeled polyclonal antibodies obtained from white rabbit immunized with purified BVDV was used as the standard,and the sheep anti-rabbit HRP-IgG was used as the C line to assemble into a double antibody sandwich colloidal gold chromatography reagent strip and optimized the reaction conditions.The results showed that the optimal protein labeling amount was 30μg/mL,and the optimal concentration of the C and T lines was 0.5mg/mL.Serum against swine fever virus(CSFV),bovine infectious rhinotracheitis virus(IBRV)serum,bovine papular stomatitis virus(BPSV)isolate,or BVDV clinical isolate was used to evaluate the specificity of the test strip.The results showed positive results for the positive control and BVDV clinical isolate,and the rest were negative,indicating that the specificity of the test strip was strong and there was no cross-reaction.The minimum detection amount of this strip is 10^(4.6)ELD_(50)/mL.The prepared test strips were applied to 34 bovine serum samples collected in clinical practice,and the RT-PCR method was used as the control for comparison.The results showed that the positive detection rate of colloidal gold test strips was 17.5%(12/68),and the positive detection rate of RT-PCR was 26.5%(18/68).Thus,the consistency rate between the two methods was 88.24%.In this study,the double antibody-based sandwich method was used to prepare BVDV colloidal gold immunochromatography test strips for the first time in China,which was convenient and fast,with good specificity and sensitivity,which was suitable for the rapid screening of BVDV in cattle farms.It provided specific techniques for the further development of BVDV detection kits.

关 键 词：牛病毒性腹泻病毒 双抗体夹心 胶体金层析技术 检测 试纸条 

分 类 号：S852.65[农业科学—基础兽医学]