鸭腺病毒3型荧光RPA恒温快速检测方法的建立与应用  

Rapid Detection of Duck Adenovirus Type 3 Based on Isothermal Recombinase Polymerase Amplification Assay

作　　者：刘孟啸 蔡姝 刘建钗 赵中伟 孙宁 曲光刚[5] 苗立中[5] LIU Meng-xiao;CAI Shu;LIU Jian-chai;ZHAO Zhong-wei;SUN Ning;QU Guang-gang;MIAO Li-zhong(Hebei University of Engineering,School of Life Science and Food Engineering,Handan,Hebei 056038,China;Huazhong Agricultural University,Faculty of Animal Science and Technology、College of Veterinary Medicine,Wuhan 430070,China;Shandong Lvdu Biotechnology Co.,Ltd.,Binzhou,Shandong 256600,China;BinzhouBeizhen Middle School,Binzhou,Shandong 256200,China;Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou,Shandong 256600,China)

机构地区：[1]河北工程大学,生命科学与食品工程学院,河北邯郸056038 [2]华中农业大学,动物科技技术学院、动物医学院,武汉430070 [3]山东绿都生物科技有限公司,山东滨州256600 [4]山东省北镇中学,山东滨州256200 [5]山东省滨州畜牧兽医研究院,山东滨州256600

出　　处：《中国兽药杂志》2023年第11期8-15,共8页Chinese Journal of Veterinary Drug

基　　金：“十四五”国家重点研发计划项目子课题“重要人兽共患病恒温快检测试剂盒的研制和注册”(2022YFC2304002-3)。

摘　　要：鸭腺病毒3型(Duck adenovirus type 3,DAdV-3)是我国近年来新发的禽腺病毒,致病力较强,已经严重影响到鸭养殖业。为实现对DAdV-3的快速检测,以DAdV-3的Hexon基因为靶基因,设计特异性重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)引物和探针,并构建重组标准质粒命名为DAdV-H-18T,优化RPA反应条件后,建立DAdV-3的荧光RPA恒温快速检测方法。结果显示,该检测方法最佳反应条件为38℃、18 min;特异性强,与其他常见鸭病病原如鸭肝炎病毒、鸭坦不苏病毒等均无交叉反应。对重组质粒DAdV-H-18T的最低检测限为1 copy/μL;采用建立的RPA检测方法和qPCR对55份鸭组织临床样本进行检测,阳性率分别为65.4%、50.9%,检测符合率为92.7%。研究建立的DAdV-3荧光RPA方法,检测速度快,敏感性高,特异性强,为该病临床快速检测提供了有力工具。Duck adenovirus type 3(DAdV-3)is a newly discovered poultry adenovirus in my country in recent years.It has a strong pathogenicity and has seriously affected the duck breeding industry.In order to realize the rapid detection of DAdV-3,specific Recombinase polymerase amplification(RPA)primers and probes were designed with the Hexon gene of DAdV-3 as the target gene,and the recombinant standard plasmid was constructed and named DAdV-H-18T.After optimizing the RPA reaction conditions,the establishment Fluorescent RPA isothermal rapid detection method for DAdV-3.The results showed that the optimal reaction condition of the detection method was 38℃、18 minutes;the specificity was strong,and there was no cross-reaction with other common duck disease pathogens such as duck hepatitis virus and duck tambusu virus.The minimum detection limit of the recombinant plasmid DAdV-H-18T was 1 copy/μL;the established RPA detection method and qPCR were tested on 55 duck tissue clinical samples,and the results showed that the positive rates of RPA and qPCR were 65.4%,50.9%,the detection coincidence rate was 92.7%.This study successfully established the fluorescent RPA method of DAdV-3,which has the advantages of fast speed,high sensitivity and strong specificity,and provides a powerful tool for the rapid clinical detection of the disease.

关 键 词：DAdV-3 Hexon基因 RPA 恒温快速检测 

分 类 号：S852.65[农业科学—基础兽医学]