检测牛肠道病毒RT-RAA-LFD方法的建立与初步应用  

Establishment and preliminary application of RT-RAA-LFD method for the detection of bovine enteroviruses

作　　者：张福慧 郑学博 崔续媛 章凡 张芷源 胡俊英 张群 王新平 ZHANG Fuhui;ZHENG Xuebo;GUI Xuyuan;ZHANG Fan;ZHANG Zhiyuan;HU Junying;ZHANG Qun;WANG Xinping(College of Veterinary Medicine,Jilin University,Changchun 130062,China;State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]吉林大学人畜共患传染病重症诊治全国重点实验室,吉林长春130062

出　　处：《中国兽医学报》2024年第11期2348-2355,共8页Chinese Journal of Veterinary Science

基　　金：“十三五”国家重点研发计划资助项目(2017YFD0500104,2016YFD0500904)

摘　　要：本研究研发了一种基于重组酶介导核酸扩增(RAA)技术结合胶体金试纸条快速检测牛肠道病毒(BEV)的方法。以高度保守的BEV 5′UTR作为靶序列,设计出特异性引物,下游引物5′端标记生物素,探针5′端标记6-FAM,初步建立RT-RAA方法。将鼠源抗6-FAM单克隆抗体作为金标抗体,链霉亲和素包被于检测线,羊抗鼠IgG包被于质控线,组装试纸条。将RAA技术与制备的胶体金试纸条相结合,建立起检测牛肠道病毒的RT-RAA-LFD方法,并对该方法的特异性、敏感性与重复性及临床应用等方面进行了评价。结果显示,该方法最佳引物浓度为5μmol/L,在35℃反应8 min即可完成对BEV核酸的扩增;该方法最低检测限为10^(1)拷贝/μL,且与牛病毒性腹泻病毒、牛细小病毒、口蹄疫病毒均无交叉反应;制备的试纸条于4℃条件下,有效期至少为90 d;74份临床样品检测结果显示,该方法与RT-PCR检测结果一致。上述结果表明,本研究建立的BEV RT-RAA-LFD方法灵敏度高、特异性强,且操作更加便捷,适用于临床现场检测,为BEV感染的诊断和流行病学调查提供了新的技术手段。A recombinant enzyme-mediated nucleic acid amplification(RAA)technology combined with colloidal gold test strips was developed for the rapid detection of bovine enterovirus(BEV).Using the highly conserved BEV 5'UTR as the target sequence,the primers were designed and screened.Downstream primer labeled with biotin at the 5'end and the probe labeled with 6-FAM at the 5'end were used to establish the RT-RAA method.The test strips were assembled by using mouse-derived anti-6-FAM monoclonal antibody as the gold standard antibody,with a streptavidin encapsulated in the detection line and sheep anti-mouse IgG encapsulated in the quality control line.A RT-RAA-LFD method was established by combing RAA technique with the prepared lateral flow device test strips for the detection of bovine enterovirus nucleic acids.The specificity,sensitivity,repeatability,and clinical application of the method are also evaluated.The results showed that the optimal primer concentration of this method was 5μmol/Land the amplification of BEV nucleic acids was accomplished by reacting at 35℃for 8 min with the lowest detection limit of 10^(1)copies/μL.No cross-reactivity with bovine viral diarrhea virus,bovine parvovirus,and foot-andmouth disease virus was observed.The efficacy for the prepared test strips was at least for 90 d kept ar 4℃.Detection of 74 clinical samples yielded a similar result compared with RT-PCR method.The above results demonstrated that the BEV RT-RAA-LFD method established in this study has high sensitivity,specificity,and more convenient to use,which is suitable for clinical detection on-site and provides a new technical tool for the diagnosis and epidemiological investigation of BEV infection.

关 键 词：牛肠道病毒 5′UTR RT-RAA-LFD 胶体金试纸条 

分 类 号：S852.65[农业科学—基础兽医学]