重组酶介导等温核酸扩增结合核酸试纸条即时检测猪流行性腹泻病毒方法的建立  

Establish of A Rapid and Visible Assay Method for Detection of Porcine Epidemic Diarrhea Virus Using Recombinase-aided Isothermal Nucleic Acid Amplification in Combination with Nucleic Acid Strips

作　　者：易玮婕 李嘉豪 赵伊然 单衍可 刘斐[1] YI Weijie;LI Jiahao;ZHAO Yiran;SHAN Yanke;LIU Fei(College of Veterinary Medicine,Nanjing Agricultural University,Single Molecule Nanometry Laboratory(Sinmolab),Nanjing 210095 China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学动物医学院单分子纳米实验室,南京210095 [2]江苏省农业科学院兽医研究所,南京210014

出　　处：《病毒学报》2024年第5期1054-1061,共8页Chinese Journal of Virology

基　　金：湖南省重点研发项目(项目号:2023NK2017),题目:畜禽疫病诊断及疫苗免疫评价技术研究与示范。

摘　　要：本文旨在建立一种基于重组酶介导等温扩增技术(Recombinase aided amplification,RAA)结合核酸试纸条的猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)可视化检测方法,以应用于养殖场内部实现现场快速检测。选择PEDV N基因保守序列为模板,设计特异性引物及荧光探针,对引物进行对比筛选,对反应条件、引物浓度、探针浓度进行优化,并对其特异性、敏感性和重复性进行评价。将建立的检测方法应用于临床样品,并与荧光定量PCR方法进行比较。结果表明:建立的方法在38℃恒温10 min扩增反应以及5 min侧流向层析后,即可完成检测,与猪繁殖与呼吸综合征病毒、猪瘟病毒、伪狂犬病毒、猪圆环病毒2型、猪德尔塔冠状病毒、猪轮状病毒、猪传染性胃肠炎病毒均无交叉反应,最低检测限可达101拷贝/μL。应用该方法检测20份阴性和20份阳性,共40份临床样本,检测结果与荧光定量PCR方法保持一致。本研究建立的RAA结合核酸试纸条的方法具有特异性好、灵敏度高、耗时短、仪器设备简便等优点,适用于基层现场快速检测。The aim of this study was to establish a visualization detection method for porcine epidemic diarrhea virus(PEDV)based on recombinase-mediated isothermal amplification(RAA)technology combined with nucleic acid strip tests,which can be applied to on-site rapid detection in breeding farms.The conserved sequence of the PEDV N gene was selected as template.Specific primers and fluorescent probes were designed,and the primer sets were compared for screening.The reaction conditions,primer concentration,and probe concentration were optimized,and the specificity,sensitivity,and repeatability were evaluated.The established detection method was applied to clinical samples and compared with quantitative fluorescent PCR.Results showed that the established method could complete the detection in 10 minutes at an isothermal temperature of 38°C,plus 5 minutes of lateral flow strip testing.There was no cross-reactivity with porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV),porcine circovirus 2(PCV2),porcine deltacoronavirus(PDCoV),porcine rotavirus(PoRV),transmissible gastroenteritis of swine virus(TGEV).The lowest detection limit was 101 copies/μL.The detection results were consistent with those of the quantitative fluorescent PCR in detection of 40 clinical samples,including 20negative and 20 positive samples.The method established in this study has advantages of high specificity,sensitivity,short testing time,and simple instrumentation,making it suitable for on-site testing in grassroots areas.

关 键 词：猪流行性腹泻病毒 重组酶介导等温核酸扩增技术 现场快速检测 

分 类 号：S852.65[农业科学—基础兽医学]