塞尼卡谷病毒重组酶聚合酶扩增技术(RPA)实时荧光检测方法的建立  

Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification(RPA) for the Detection of Seneca Valley Virus

作　　者：樊晓旭[1] 哈登楚日亚 王英丽[1] 王姣 刘春菊[1] 迟田英[1] 赵永刚[1] 张志诚[1] 吴晓东[1] 王志亮[1] Fan Xiaoxu;Hadengchuriya;Wang Yingli;Wang Jiao;Liu Chunju;Chi Tianying;Zhao Yonggang;Zhang Zhicheng;Wu Xiaodong;Wang Zhiliang(National Research Center for Exotic Animal Disease,China Animal Health and Epidemiology Center,Qingdao,Shandong 266032;College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018;Changzhou Animal Disease Prevention and Control Center,Changzhou,Jiangsu 213002)

机构地区：[1]中国动物卫生与流行病学中心,国家外来病研究中心,山东青岛266032 [2]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [3]常州市动物疫病预防控制中心,江苏常州213002

出　　处：《中国动物检疫》2017年第8期81-86,共6页China Animal Health Inspection

基　　金：国家重点研发计划(2016YFD0501104)

摘　　要：塞尼卡谷病毒(Seneca valley virus,SVV)可感染猪,引起类似口蹄疫病毒感染造成的水疱性病变,新生仔猪病死率达30%~70%。本研究利用重组酶聚合酶扩增技术(RPA),针对SVV 3D基因设计了引物和探针,并进行筛选、反应条件优化以及敏感性、特异性和重复性试验,建立了实时荧光RPA方法。结果显示,本方法在40℃、10 min内检测的最低浓度为28拷贝/μL;与口蹄疫病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒无交叉反应;通过3次重复试验检测2.8×10~6~2.8×10~1拷贝/μL的6个稀释度样品,在荧光强度达1500 mV所需时间变异系数范围在2.44%~14.95%。该等温、快速扩增方法为猪群出现水疱性疾病的鉴别诊断提供了技术支持,对及时采取适当防控措施具有重要意义。Seneca valley virus(SVV)can affected swine,which can cause vesicular lesions similar to the infectionof foot and mouth disease virus,with the neonatal mortality rate of30%~70%.A real-time RPA method was establishedin this study,based on recombinase polymerase amplification technology(RPA).Assemblies of primers and probestargeting SVV3D gene were developed and screened,while reaction conditions were optimized before sensitivity,specificity and repeatability of the test were determined.The results showed that the lowest concentration of28copies/μLcould be detected within10min at40℃.No cross reaction was found between SVV and the foot and mouth diseasevirus,classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrheavirus and transmissible gastroenteritis virus.In the3repeated tests of six series diluted samples(2.8×106~2.8×101copies/μL),the coefficient of variation of time when the fluorescence intensity reached1500mV was in the range of2.44%~14.95%.The isothermal and rapid amplification method provides technical support for the differential diagnosisof vesicular disease among swine,and it is of great significance to take appropriate preventive measures in a timelymanner.

关 键 词：塞尼卡谷病毒 实时 荧光 重组酶聚合酶扩增 

分 类 号：S852.65[农业科学—基础兽医学]