摘要
自2007年在猪粪便中首次检测到猪嵴病毒(PKV)以来,该病毒已在世界范围内被广泛报道,并可能与仔猪的腹泻有关。本研究首次建立了以重组酶聚合酶扩增结合侧流层析试纸条(RPA-LFD)的技术来检测PKV的快速、可视化的诊断方法,并对此方法的敏感性、特异性及重复性进行了评价。结果显示,该方法具有良好的特异性,仅可以特异性扩增PKV,与其它主要猪病毒没有交叉反应;最佳反应时间和温度是RPA在37℃下20 min及LFD在室温下10 min;检测极限为1×10~2拷贝的PKV。利用本研究建立的RPA-LFD方法和常规RT-PCR方法同时检测20份猪的临床粪便样品,结果显示,RPA-LFD方法检测猪嵴病毒阳性率为60%,明显高于常规RT-PCR的50%。上述结果表明,RPA-LFD方法可应用于猪嵴病毒的快速检测。
Since porcine kobuvirus(PKV)was first detected in fecal samples from pigs in 2007,this virus has been frequently reported worldwide and is associated with diarrhea in piglets.In the present study,a rapid and visual diagnostic method was first developed using recombinase polymerase amplification combined with a lateral flow dipstick(RPA-LFD)to detect PKV,and the sensitivity,specificity and repeatability of this method were evaluated.In result,the RPA-LFD assay could specifically amplify PKV and exhibited no cross-amplification of other virus;the optimized reaction time and temperature for this assay were 20 min at 37℃for RPA followed by 10 min at room temperature for the LFD.The detection limit of the RPA-LFD assay was 1×10~2 copies of PKV.Besides20 pig stool sampleswere detected using RPA-LFD assay and the results showed that the positive rate of porcine kobuvirus was(60%)and significantly higher than that detected by the conventional RT-PCR(50%).According to the above results,the developed RPA-LFD assaycan be applied to clinical diagnosis and rapid detection of porcine kobuvirus.
作者
蔡应奎
刘新生
张丽萍
周鹏
王永录
CAI Ying-kui;LIU Xin-sheng;ZHANG Li-ping;ZHOU Peng;WANG Yong-lu(Gansu Yasheng Agricultural Research Institute Co.LTD.,Lanzhou 730010,China;State Key Labotorary of Veterinary Etiological Biology/Lanzhou Veterinary Reasearch Insttitute,Chinese Academy of Argricultural Science,Lanzhou 730046,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2020年第7期820-824,共5页
Chinese Veterinary Science
基金
国家生猪产业体系项目(CARS-35)
“十三五”国家重点研发计划项目(2016YFD0501505)。
