摘要
为开发一种可靠、简易、便捷的现场猪流行性腹泻病毒(PEDV)检测方法,基于反转录重组酶介导等温扩增(RT-RAA)和侧向流试纸条(LFD)技术,建立了用于现场检测PEDV的RT-RAA-LFD方法。根据PEDV N基因保守序列进行引物和探针设计,通过试验优选出一套引物及一条探针,同时对RT-RAA反应时间和温度条件进行优化。优化后的方法在37℃恒温反应15 min,即可实现对PEDV目的基因片段的有效扩增;与其他常见腹泻病毒无交叉反应,最低检测限为1.23×10^(1) copies/μL;对134份临床样品进行检测,阳性检出率为5.97%,与国标检测方法结果一致。结果表明,本研究建立的PEDV RT-RAA-LFD检测方法操作简便,结果特异、灵敏,不需要专业的实验室设备和实验人员,适用于在资源有限地区对PEDV的检测,能够满足基层临床工作人员的需求。
In order to develop a reliable,simple and convenient method for on-site detection of porcine epidemic diarrhea virus(PEDV),an RT-RAA-LFD assay based on reverse transcription recombinase-aided isothermal ampliff cation(RT-RAA)and lateral ff ow dipstick(LFD)was established for such purpose.Primers and probes were designed based on the conserved sequence of PEDV N gene,and a set of optimal primers and a probe were tested and selected.The reaction time and temperature conditions of RT-RAA were also optimized.Target PEDV gene fragments were eff ectively ampliff ed by the optimized method after a 15-minute isothermal reaction at 37°C;there was no cross-reactivity with other common diarrheal viruses,with a limit of detection(LOD)of 1.23×10^(1) copies/μL;based on 134 clinical samples,the positive detection rate was 5.97%,which was consistent with that obtained by the national standard.In conclusion,the RT-RAA-LFD established in the study was user-friendly,speciff c and sensitive,making it suitable for PEDV detection in resource-limited areas as required by local clinical staff,as it could work without any specialized laboratory equipment or trained personnel.
作者
仇德洋
王海燕
郑佳
杜建才
陈昭
李桂梅
Qiu Deyang;Wang Haiyan;Zheng Jia;Du Jiancai;Chen Zhao;Li Guimei(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,Shandong,China;Center for Drug Safety Evaluation of Lunan Pharmaceutical Group Corporation,Linyi 273499,Shandong,China;Dezhou Animal Husbandry and Veterinary Aff airs Development Center,Dezhou 253000,Shandong,China)
出处
《中国动物检疫》
2025年第7期90-95,共6页
China Animal Health Inspection
基金
山东省生猪产业技术体系项目(SDAIT-08-07)。
