猪瘟病毒野毒株与疫苗株E2基因双重TaqMan real-time PCR鉴别检测方法的建立  

Detection of E2 gene from classical swine fever virus using TaqMan quantitative real-time PCR technology and expression of E2 protein by E. coli

作　　者：杜冬华 薛拥志[1] 王爱华[1] 周静[1] 孙全文[1] 邱殿锐 郭建军 

机构地区：[1]河北北方学院动物科技学院,河北张家口075131 [2]承德市畜牧研究所,河北承德067000

出　　处：《中国兽医学报》2015年第12期1898-1902,共5页Chinese Journal of Veterinary Science

基　　金：河北北方学院重大课题资助项目(ZD201307);河北省2014年科技攻关资助项目(14226604D);河北省张家口市科学技术和地震局资助项目(1411060C)

摘　　要：为建立猪瘟病毒（CSFV）野毒株和疫苗株快速定量鉴别检测方法，根据CSFV野毒株和疫苗株E2基因序列差异，设计了2对特异性引物及1条TaqMan探针，以含CSFV HB株和C-株E2基因的重组质粒pBS-E2C和pBS-E2作为标准品，建立了能同时检测CSFV野毒株和疫苗株的双重TaqMan real-time PCR鉴别检测方法。结果表明，建立的CSFV双重TaqMan real-time PCR标准曲线Ct值与1×10^1-l×10^6 copies／μL，之间的E2基因拷贝数呈良好的线性关系，灵敏度达10copies／μL，且特异性和重复性很好。综上，本试验建立的TaqMan real-time PCR检测方法可用于CSFV野毒株和疫苗株的鉴别诊断及病原定量分析。This experiment was conducted to establish a duplex TaqMan real-time PCR for differen- tiating C-strain vaccine and wild type viruses of CSFV. According to the differences of the E2 gene sequences between C-strain and wild type of CSFV, two pairs of specific primers and a TaqMan probes were designed in the E2 gene. The recombinant plasmid, which contained E2 gene of C- strain vaccine and wild-type viruses of CSFV was used to establish melting and standard curves of the duplex TaqMan quantitative real time PCR. The standard curve for the viral genomic copy number and threshold cycle ranging from 1×10^1 1×10^6 copies/μL E2 gene were linear. Sensitivity of the method was 10 copies,with a good specificity and repeatability. These data indicated that the duplex TaqMan quantitative real-time PCR can be used to CSFV diagnosis and quantification.

关 键 词：猪瘟病毒 双重荧光定量PCR TAQMAN探针 野毒株 疫苗株 

分 类 号：S852.65[农业科学—基础兽医学]