摘要
为针对传染性造血器官坏死病病毒(IHNV)建立一种可实现现场快速、准确的检测方法,结合重组酶介导扩增(RAA)和侧向流试纸条(LFD)技术,在IHNVG基因保守区域设计引物和探针,通过优化反应时间和温度等条件,建立了可用于IHNV现场可视化检测的RT-RAA-LFD方法。优化后的检测方法在35℃水浴锅中恒温反应15 min即可实现对IHNV目的基因片段的有效扩增。结果显示:该方法可以特异性地检测出IHNV,不与其他常见鱼类病毒发生交叉反应;对IHNV质粒标准品的检测下限可以达到2.15×101 copies/μL;采用该方法和实时荧光RT-PCR、实时荧光RT-RAA同时对35份临床样本进行检测,其阳性符合率为100%。上述结果表明,本研究针对IHNV成功建立了一种快速、准确、便捷的试纸条检测方法,并可适应于多种场合。
To establish a rapid and accurate method for detection of infectious haematopoietic necrosis virus(IHNV),primers and probes were designed according to the conserved sequence of IHNV G gene.A real-time recombinase-aid amplification combined with lateral flow dipstick(RT-RAA-LFD)method was established for visual detection of IHNV in the field with a water bath within 15 min at 35℃after being optimized.The assay was specific with the target sequence of IHNV with a detection limit up to 2.15×101 copies/μL.Thirty-five clinical samples were tested by this method,showing 100%consistency with the real-time RT-PCR and RT-RAA.The RT-RAA-LFD assay developed in this study was a rapid,reliable and simple method which can be broadly used in the farms and diagnostic on the samples with suscepected clinical signs.
作者
廖立珊
徐彬
陈兵
吴江
王津津
朱裕敏
刘荭
王婉君
孙洁
LIAO Li-shan;XU Bin;CHEN Bing;WU Jiang;WANG Jin-jin;ZHU Yu-min;LIU Hong;WANG Wan-jun;SUN Jie(Animal and Plant Inspection and Quarantine Technology Center,Shenzhen Customs,Shenzhen 518045,China;Shenzhen Institute of Quality&Safety Inspection and Research,Shenzhen 518000,China;Dalian Ocean University,Dalian 116023,China;Shenzhen Branch,China Resources WuFeng Meat Products(Henan)Co.,Ltd.,Shenzhen 518031,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第8期991-996,共6页
Chinese Veterinary Science
基金
深圳市可持续发展项目(KCXFZ20211020165547010)
广东省“生物安全技术”专项重点研发项目(2022B1111030001)。
关键词
传染性造血器官坏死病病毒
重组酶介导扩增
侧向流试纸条
快速检测
infectious haematopoietic necrosis virus
recombinase-aid amplification
lateral flow dipstick
rapid detection