北美型猪繁殖与呼吸综合征病毒免疫层析试纸条的研制及初步应用  

Development and preliminary application of immunochromatography strip for detecting North American-type porcine reproductive and respiratory syndrome virus

作　　者：李宛生 李敏华 张洪亮[1] 李超 许浒 付军 龚帮俊 郭镇洋 刘建波[1] 彭金美[1] 周国辉[1] 王倩[1] 田志军[1] LI Wan-sheng;LI Min-hua;ZHANG Hong-liang;LI Chao;XU Hu;FU Jun;GONG Bang-jun;GUO Zhen-yang;LIU Jian-bo;PENG Jin-mei;ZHOU Guo-hui;WANG Qian;TIAN Zhi-jun(National Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Beijing IDEXX Yuanheng Laboratories,Co.,Ltd,Beijing 101318,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,黑龙江哈尔滨150069 [2]北京爱德士元亨生物科技有限公司,北京101318

出　　处：《中国预防兽医学报》2022年第8期842-849,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金优秀青年项目(YQ2019C032);兽医生物技术重点实验室自主研究课题(SKLVBP202122)。

摘　　要：为研制一种快速检测北美型猪繁殖与呼吸综合征病毒(PRRSV)的免疫层析试纸条(ICS),本研究以红色乳胶微球偶联的PRRSV N蛋白单克隆抗体(MAb)1H4为检测抗体,并包被在结合垫上,以MAb 1H4和羊抗鼠IgG为捕获抗体,并分别包被在硝酸纤维素膜上作为检测线和质控线,经各反应条件的优化建立了检测PRRSV的乳胶微球ICS。各反应条件优化结果显示,乳胶微球偶联的MAb即检测抗体浓度为1.5 mg/mL,检测线MAb即检测线捕获抗体的浓度为2.0 mg/mL,质控线羊抗鼠IgG即质控线捕获抗体的浓度为1.0 mg/mL。特异性试验结果显示,该方法可特异性检测我国出现的高致病性、NADC30-like、NADC34-like等北美型PRRSV代表株,与欧洲型PRRSV DV疫苗株及猪的其他常见病毒(CSFV、PRV、PCV2、PEDV、TGEV、PPV)均无交叉反应;敏感性试验结果显示,该方法对系列稀释PRRSV(10^(5.0) TCID_(50)/mL~2.5×10^(2.0) TCID_(50)/mL)的最低检测限为5×10^(2.0) TCID_(50)/mL,对2倍倍比稀释的重组N蛋白(rN,24000 ng/mL~3.75 ng/mL)的最低检测限为15 ng/mL;重复性试验结果显示,同一批次和不同批次试纸条的检测结果均无差异;对不同类型北美型PRRSV株(高致病性株、NADC30-like株、类高致病性株)感染的猪血清检测结果显示,利用该ICS可以从相应猪血清中检测到不同北美型的PRRSV。对采集的108份疑似PRRSV感染的猪肺组织及血清样品分别采用本研究建立的ICS及本研究室建立的PCR方法检测,结果显示,ICS检测的阳性率为37.03%(40/108),PCR检测的阳性率为42.59%(46/108),二者的总体符合率为83.33%。上述结果表明,该方法可以用于我国PRRSV主要流行株及临床样品的检测。本研究首次以同一株MAb分别作为检测抗体和检测线捕获抗体,建立检测PRRSV的乳胶微球ICS,为现地PRRSV流行病学调查及临床快速检测提供了新的技术手段。In order to develop an immunochromatography strip(ICS)for rapid detection of North American-type porcine reproductive and respiratory syndrome virus(PRRSV),this study used the red latex microspheres coupled with anti-PRRSV N protein MAb 1H4 as the detection antibody,which was coated on the conjugate pad.In addition,MAb 1H4 and goat anti-mouse IgG antibodies as the capture antibodies were coated on the nitrocellulose membrane as the test and control lines,respectively.The ICS for detecting PRRSV based on the latex microsphere was established by optimizing the reaction conditions.The optimized reaction condition showed that the optimal concentration of MAb coupled with latex microspheres(detection antibody)was 1.5mg/mL,the optimal concentration of MAb(detection line capture antibody)in the test line was 2.0mg/mL,and the optimal concentration of goat anti-mouse IgG(quality control line capture antibody)in the control line was 1.0mg/mL.The specificity test showed that North American-type PRRSV representative strains in China,such as high pathogenic strain,NADC30-like strain,and NADC34-like strain,could be specifically detected,and no cross-reaction with European PRRSV vaccine DV strain or other viruses commonly infect pigs(CSFV,PRV,PCV2,PEDV,TGEV,PPV).The sensitivity test showed that the minimum detection limit of PRRSV was 5×10^(2.0)TCID_(50)/mL,and that of recombinant N(rN)protein was 15ng/mL.The repeatability test demonstrated that there was no difference between the same batches and the different batches of strips.The detection results for the sera that were collected from different North American-type PRRSV strains(high pathogenic strain,NADC30-like strain,and high-pathogenic-like strain)infecting pigs showed that these different types of PRRSV can be detected using this ICS.One hundred and eight samples from lung tissues and serum suspicious of PRRSV infection were tested using the established ICS and PCR,respectively.The results showed that the positive rate of the ICS test was 37.03%(40/68),and that of the P

关 键 词：猪繁殖与呼吸综合征病毒 北美型 N蛋白 免疫层析试纸条 

分 类 号：S852.65[农业科学—基础兽医学]