伪狂犬病病毒野毒和疫苗毒双重RPA鉴别检测方法的建立和应用  

Development and application of the dual RPA differential detection method for wild virus and vaccine strain of pseudorabies virus

作　　者：刘立兵 王建昌[1,2] 耿云云 王金凤[1,2] 袁万哲 LIU Li-bing;WANG Jian-chang;GENG Yun-yun;WANG Jin-feng;YUAN Wan-zhe(Hebei Academy of Inspection and Quarantine, Shijiazhuang 050051, China;Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China;College of Life Scinece, Hebei Normal University, Shijiazhuang 050024, China;College of Veterinary Medicine, Agricultural University of Hebei, Banding 071001, China)

机构地区：[1]河北省检验检疫科学技术研究院,河北石家庄050051 [2]河北出入境检验检疫局技术中心,河北石家庄050051 [3]河北师范大学生命科学学院,河北石家庄050024 [4]河北农业大学动物医学院,河北保定071001

出　　处：《中国预防兽医学报》2018年第4期306-310,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省自然科学基金青年项目(C2017325001);河北省科技攻关计划(16226604D)

摘　　要：为建立伪狂犬病病毒(PRV)野毒和疫苗毒的快速鉴别检测方法,本研究基于PRV gB和gE基因保守区域设计引物,建立了一种双重重组酶聚合酶扩增方法(RPA)。本研究所建立的双重RPA方法在38℃水浴锅中恒温反应20min,能够在同一个反应体系中实现对PRV野毒和疫苗毒的特异性鉴别检测,而与其它常见的猪病毒没有交叉反应。所建立的双重RPA方法对PRV gB和gE基因的检测限均为102拷贝,并且与荧光定量PCR方法检测限一致。通过对4株不同PRV株和37份临床样品的检测,结果表明双重RPA方法能够实现对不同PRV株的检测,对临床样品中PRV gB和gE基因的检出率均为45.9%(17/37),与荧光定量PCR方法检测的符合率为100%。本研究所建立的双重RPA方法反应快速、操作简便、结果可靠,可有效应用于对PRV野毒和疫苗毒的快速鉴别检测。In this study, a rapid method for detection and identification of pseudorabies virus （PRV） wild strain and gE-deleted PRV vaccine strain was developed using dual recombinase polymerase amplification （RPA） based on the primers designed according to the conservative region of PRV gB and gE genes. With this method, the wild and gE-deleted PRV vaccine could be specifically detected and differentiated in one single reaction system which takes 20 minites in 38 ℃water bath, without cross-reactions with other non-targeted swine viruses. The detection limit for gB and gE genes both was 10^2 copies in this dual RPA assay, which was accord with the real-time PCR test results. Four different PRV strains were effectively detected and identified by this dual RPA method, and the detection rate for gB and gE genes from 37 clinical samples was 45.9% （17/37）, which demonstrated a 100% diagnostic agreement with the real-time PCR. The dual RPA assay established in this researchprovides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV,

关 键 词：伪狂犬病病毒 等温扩增 聚合酶重组酶扩增 鉴别检测 

分 类 号：S852.65[农业科学—基础兽医学]