摘要
旨在建立一种快速现场筛查非洲猪瘟病毒(ASFV)基因缺失株的方法,以便鉴别我国复杂的ASFV感染情况。通过分析国内ASFV流行态势,选择MGF-505R基因及B646L基因,设计特异性引物及exo探针,对其进行敏感性、特异性分析,并进行临床应用。利用双重-重组酶聚合酶扩增(RPA)方法检测ASFV MGF-505R及B646L基因,结果显示:检测时间在15 min内,最低检测限均为10 copies/反应,且与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型等均无交叉反应;利用所建立方法对266份临床样品进行检测,结果与世界动物卫生组织(WOAH)推荐荧光PCR检测方法的符合率为100%。建立的ASFV MGF-505R基因缺失株的荧光RPA检测方法适配可移动式荧光RPA检测仪,具有快速、灵敏度高、特异性强的优点,适用于ASFV现场快速临床检测,为非洲猪瘟疫情防控提供一种新的筛查手段。
This study was to establish a rapid field screening method for screening African swine fever(ASF) gene-deleted mutants in order to identify the complex infection situation of ASF in China.Based on the epidemic trends of ASFV in China,the specific primers and exo probes were designed and synthesized by analyzing the conserved regions of the ASFV B646L and MGF-505R genes,in order to establish a double real-time RPA method for detection of the above-mentioned mutants.The sensitivity and specificity of the method were analyzed.In addition,the method was tested in clinical application using the optimized RPA.The result indicated that the double real-time RPA method could monitor the detection process within 15 min.Its minimum detection of RPA was up to 10 copies/response for both the genes.The method was highly specific,without cross-reactions with classical swine fever virus,porcine respiratory and reproductive syndrome virus,pseudorabies virus,porcine parvovirus and porcine circovirus type 2.Then,266 clinical samples were tested using the dual real-time RPA and WOAH recommended real-time PCR,with a coincidence rate of up to 100%.The double real-time RPA method for detecting ASFV MGF-505R gene-deleted mutants could be adapted to the mobile real-time RPA detector,which possessed the advantages of rapidity,high sensitivity and strong specificity.The method is applicable for rapid clinical detection of ASFV,and it could serve as a new screening method for prevention and control of ASF.
作者
邓俊花
吕继洲
陈冬杰
袁向芬
魏方
赵文军
吴绍强
DENG Junhua;LÜJizhou;CHEN Dongjie;YUAN Xiangfen;WEI Fang;ZHAO Wenjun;WU Shaoqiang(Chinese Academy of Inspection and Quarantine,Beijing 100176,China;CAIQ Center for Biosafety,Sanya 572025,China)
出处
《畜牧与兽医》
CAS
北大核心
2023年第12期85-90,共6页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划项目(2022YFD1800605)。
