鲤春病毒血症病毒RT-RAA-LFD检测方法的建立  

Development of a RT-RAA-LFD assay for the detection of Spring viremia of carp virus

作　　者：朱裕敏 吴江 廖立珊 王婉君 孙洁 陈兵 王津津 郑晓聪 贾鹏 刘荭 ZHU Yu-min;WU Jiang;LIAO Li-shan;WANG Wan-jun;SUN Jie;CHEN Bing;WANG Jin-jin;ZHENG Xiao-cong;JIA Peng;LIU Hong(Dalian Ocean University,Dalian 116023,China;Animal and Plant Inspection and Quarantine Technology Center,Shenzhen Customs,Shenzhen 518054,China;China Resources WuFeng Meat Products(Henan)Co.,Ltd.Shenzhen Branch,Shenzhen 518172,China;Shenzhen Technology University,Shenzhen 518118,China)

机构地区：[1]大连海洋大学,辽宁大连116023 [2]深圳海关动植物检验检疫技术中心,广东深圳518045 [3]华润五丰肉类食品(河南)有限公司深圳分公司,广东深圳518172 [4]深圳技术大学,广东深圳518118

出　　处：《中国预防兽医学报》2023年第9期916-921,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省生物安全专项(2022B1111030001);深圳市可持续项目(KCXFZ20211020165547010)。

摘　　要：为建立一种可现场快速准确检测鲤春病毒血症病毒(SVCV)的方法,本研究结合重组酶介导扩增(RAA)和侧向流试纸条(LFD)技术,根据SVCV L基因保守区域设计引物及探针,通过优化反应时间和温度等条件,初步建立了可用于SVCV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SVCV目的基因片段的有效扩增。结果显示,该方法可以特异性的检测SVCV,并且不与病毒性出血败血症病毒、传染性鲑鱼贫血症病毒、病毒性神经坏死病毒等其他常见鱼类病毒发生交叉反应;对SVCV重组质粒标准品的检测限为2.42×10^(2)拷贝/μL,并具有较好的稳定性和重复性;采用该方法和病毒分离、套式RT-PCR、荧光定量RT-PCR、荧光定量RT-RAA共5种方法同时对45份临床样品检测,其中病毒分离、套式RT-PCR和荧光定量RT-PCR均检测到29份阳性样品,荧光定量RT-RAA和本研究建立的方法均检测到28份阳性样品,本研究与前3种方法的阳性符合率为97.8%,与荧光定量RT-RAA的阳性符合率为100%。上述结果表明,本研究建立的SVCV RT-RAA-LFD检测方法简便快捷、特异性强、敏感性高、结果可靠,且不依赖于复杂的仪器,可适用于现场和实验室对SVCV的快速检测。To establish a rapid and accurate on-site detection method for Spring viraemia of carp virus(SVCV),this study combined recombinase-aid amplification(RAA)and lateral flow dipstrip(LFD)technology to detect SVCV.Primer and probes were designed according to the conserved region of the L gene,and by optimizing conditions such as reaction time and temperature,an RT-RAA-LFD method that could be used for on-site visual detection of SVCV was established.The optimized detection method can achieve effective amplification of the SVCV target gene by reacting at a temperature of 35℃in a water bath for 15 minutes.The results showed that this method can specifically detect SVCV and had no cross-reactivity with other common fish viruses such as viral hemorrhagic septicemia virus,infectious salmon anaemia virus and viral nervous necrosis virus.The detection limit of SVCV plasmid standard can reach to 2.42×10^(2) copies/μL with stable repeatability.This method and other 4 methods including virus isolation,nested RT-PCR,fluorescence quantitative RT-PCR,and fluorescence quantitative RT-RAA were used to detect 45 clinical samples at the same time.Among them,virus isolation,nested RT-PCR,and fluorescence quantitative RT-PCR detected 29 positive samples,fluorescence quantitative RT-RAA and this method detected 28 positive samples,and the positive coincidence rates were 97.8%and 100%,respectively.The above results showed that the SVCV RT-RAA-LFD detection method developed in this study was a simple,rapid,sensitive,reliable and affordable method,and it may be applied to the rapid detection of SVCV in the research laboratory and on site diagnosis.

关 键 词：鲤春病毒血症病毒 重组酶介导扩增 侧向流试纸条 恒温扩增 快速检测 

分 类 号：S852.65[农业科学—基础兽医学]