西尼罗河病毒RT-RPA检测方法的建立及初步应用  

Establishment and Applications of the RT-RPA Method for Detection of West Nile Virus

作　　者：吴江 林彦星 贾鹏 黄超华 史卫军 曹琛福 曾少灵 孙洁 阮周曦 林永涛 花群义 WU Jiang;LIN Yan-xing;JIA Peng;HUANG Chao-hua;SHI Wei-jun;CAO Chen-fu;ZENG Shao-ling;SUN Jie;RUAN Zhou-xi;LIN Yong-tao;HUA Qun-yi(Animal and Plant Inspection and Quarantine Technology Center,Shenzhen Customs,Shenzhen 518054,China)

机构地区：[1]深圳海关动植物检验检疫技术中心,广东深圳518045

出　　处：《中国兽医杂志》2021年第2期35-37,41,I0002,I0003,共6页Chinese Journal of Veterinary Medicine

基　　金：十三五国家重点研发项目(2017YFD0501805,2016YFD0500708,2018YFC0840401)。

摘　　要：为建立快速特异的西尼罗河病毒(WNV)检测方法,本研究根据WNV基因保守区域基因序列,采用Primer Explorer Version 4设计特异性的重组酶聚合酶扩增(RPA)引物和探针。经过引物筛选和条件优化,建立了在39℃恒温条件下15 min内即可完成的实时荧光RT-RPA方法。本方法能特异性检测出WNV,且与蓝舌病毒(BTV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪乙型脑炎病毒(JEV)、牛病毒性腹泻病毒(BVDV)核酸检测没有交叉反应。本方法灵敏性强,最小检出量为5.25×10^(2)拷贝/μL。采用创建的RT-RPA方法检测72份牛或猪的临床样品,结果与实时荧光定量PCR方法相符。说明本试验创建的WNV RT-RPA方法操作简便、灵敏度和特异性高,适用于WNV监测、基层实验室或出入境口岸对WNV的现场快速检测。To establish a rapid and specific method for the detection of West Nile virus(WNV),we designed specific recombinase polymerase amplification(RPA)primers and labeled probes according to the conservative genome sequences of WNV virus,using Primer Explorer Version 4 software.Through screening the primers and optimizing experimental conditions,we established the real-time RT-RPA method which can be completed within 15 min at 39℃.WNV was specifically detected by the assay without cross-reaction with other virus antigens,including BTV,PRRSV,CSFV,JEV and BVDV.The method was with high sensitivity,and its minimum detection amount was 5.25×10^(2) copies/μL.The test results of 72 bovine or pig clinical samples by the RT-RPA method and the real-time PCR method were consistent.In conclusion,the WNV RT-RPA method is simple,and has high sensitivity and specificity,which is suitable for domestic WNV monitoring,field rapid detection of WNV in grassroots laboratories or entry-exit ports.

关 键 词：西尼罗河病毒 逆转录重组酶聚合酶扩增 等温扩增 

分 类 号：R373.9[医药卫生—病原生物学]