非洲猪瘟病毒CD2v基因实时荧光PCR检测方法的建立  

Establishment of a real-time PCR assay for detection targeting the African swine fever virus CD2v gene

作　　者：王彩霞[1] 吴绍强[1] 王勤[1] 陈冬杰 张舟 冯春燕[1] 林祥梅[1] WANG Cai-xia;WU Shao-qiang;WANG Qin;CHEN Dong-jie;ZHANG Zhou;FENGChun-yan;LIN Xiang-mei(Chinese Academy of Inspection and Quarantine Sciences,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院,北京100176

出　　处：《中国兽医科学》2020年第9期1083-1089,共7页Chinese Veterinary Science

基　　金：中国检验检疫科学研究院基本科研业务费项目(2020JK020);“十三·五”国家重点研发计划项目(2016YFD0501100)。

摘　　要：为建立一种准确、特异、高效、快速的非洲猪瘟病毒(ASFV)实时荧光PCR检测方法,本研究根据ASFV CD2v基因序列,设计了TaqMan荧光PCR引物及探针,通过优化退火温度、引物及探针的比例,建立了ASFV的TaqMan实时荧光PCR检测方法。结果表明,本研究所建立的荧光PCR方法具有良好的特异性和敏感性,以重组质粒为标准品建立的TaqMan荧光PCR方法的标准曲线具有良好的线性关系,相关系数为0.988,斜率为-3.025 4,最低检测限可达到10 copies/μL,且与伪狂犬病病毒、猪细小病毒、猪圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒等多种猪病病毒不存在交叉反应。该方法重复性良好,批内和批间变异系数均小于2%。综上,本研究建立的非洲猪瘟CD2v基因实时荧光定量PCR方法为ASFV的检测提供了一种新的敏感、特异的分子检测技术。In order to establish an accurate,specific,efficient and rapid real-time fluorescent PCR detection method for African swine fever virus(ASFV),this study designed Taq Man real-time fluorescent PCR primers and probes based on the sequence of ASFV CD2v gene.By optimizing the annealing temperature,the ratio of primers and probe,the Taq Man real-time PCR method for rapid detection of ASFV was established.The results showed that the real-time fluorescent PCR method established in this study was highly sensitive and specific,and the standard curve of the Taq Man real-time fluorescent PCR method established with the constructed recombinant plasmids as a standard has a good linear relationship.The correlation coefficient is 0.988,the slope is-3.025 4,and the minimum detection limit can reach 10 copies/μL.In addition,there is no cross-reaction with various pathogens such as pseudorabies virus,porcine parvovirus,porcine circovirus type 2,classical swine fever virus and porcine reproductive and respiratory syndrome virus.This method has good repeatability was good,and the intra-and inter-assay coefficient of variation is less than 2 percent.In summary,the real-time fluorescent quantitative PCR assay for ASFV CD2v gene established in this study provides a novel,sensitive and specific molecular detection technique for ASFV detection.

关 键 词：非洲猪瘟病毒 实时荧光PCR CD2v基因 检测 

分 类 号：S852.659.1[农业科学—基础兽医学]