鸡传染性贫血病毒实时荧光RPA快速检测方法的建立  

Establishment of a Real-time Fluorescence RPA Method for Rapid Detection of Chicken Infectious Anemia Virus

作　　者：王冷眉 万培伟[1] 韩旭 易松强 闫莹莹 林翠 WANG Lengmei;WAN Peiwei;HAN Xu;YI Songqiang;YAN Yingying;LIN Cui(Jiangxi Biotech Vocational College,Nanchang,Jiangxi 330200;Jiangxi Animal Husbandry Technology Promotion Station,Nanchang,Jiangxi 330044;Institute of Animal Husbandry and Veterinary Medicine,Jiangxi Academy of Agricultural Sciences,Nanchang,Jiangxi 330200)

机构地区：[1]江西生物科技职业学院,江西南昌330200 [2]江西省农业技术推广中心,江西南昌330044 [3]江西省农业科学院畜牧兽医研究所,江西南昌330200

出　　处：《中国家禽》2025年第3期78-84,共7页China Poultry

基　　金：江西省农牧渔业科研计划项目。

摘　　要：为建立一种简便、快速的鸡传染性贫血病毒(CIAV)检测方法,试验针对CIAV VP2基因序列设计了多组引物和探针,经过引物筛选及反应温度和时间的优化,建立了CIAV的实时荧光重组酶聚合酶扩增方法(RPA),并对该方法的特异性、敏感性及重复性进行评估,应用该方法与荧光定量PCR(q PCR)同时对50份临床样品进行检测,比较两种方法的符合率。结果显示:该方法可在39℃恒温条件下20 min内快速、准确地检出CIAV,与其他6种常见的禽类病原体如传染性法氏囊病病毒(IBDV)、马立克病病毒(MDV)、J亚群禽白血病病毒(ALV-J)、新城疫病毒(NDV)、禽传染性支气管炎病毒(IBV)、传染性喉气管炎病毒(ILTV)不存在交叉反应;该方法对质粒DNA的检测限为1×10~2 copies/μL;该方法检测50份临床样品中有9份阳性,与q PCR的检测结果一致。研究表明:建立的检测CIAV的RPA操作简单、反应灵敏、特异性高、检测结果可靠,可用于鸡传染性贫血的快速检测和流行病学调查。In order to establish a simple and rapid detection method for chicken infectious anemia virus(CIAV),the multiple sets of primers and probes were designed for VP2 gene sequence of CIAV in this study.After the screening of primers and the optimization of reaction temperature and time,the detection method for CIAV real-time fluorescence recombinase polymerase amplification(RPA)were established,the specificity and sensitivity of this method were verified.The method and fluorescence quantitative PCR(qPCR)were used to detect 50 clinical samples at the same time,and the coincidence rate of the two methods was compared.The results showed that this method could quickly and accurately detect CIAV within 30 min under 39℃constant temperature,and no cross reaction with other 6 pathogens such as infectious bursal disease virus(IBDV),Marek′s disease virus(MDV),avian leukemia virus subgroup J(ALV-J),Newcastle disease virus(NDV),infectious bronchitis virus(IBV)and infectious laryngotracheitis virus(ILTV).The limit of detection for plasmid DNA by the method was 1×102 copies/μL.The 9 of 50 clinical samples were positive detected by this method,which was consistent with the results of qPCR.The study indicated that the RPA method had the advantages of simple operation,sensitive response,high specificity,reliable detection results,and could be used for rapid detection and epidemiological investigation of chicken infectious anemia.

关 键 词：鸡传染性贫血病毒 VP2基因 重组酶聚合酶等温扩增技术 快速检测 

分 类 号：S855.3[农业科学—临床兽医学]