实时荧光定量PCR检测猪细小病毒  

Detection of porcine parvovirus by quantitative real-time PCR assay

作　　者：李小康[1,2] 崔保安[1,2] 陈红英[1,2] 魏战勇[1] 郑兰兰[1,2] 赵丽[1,2] 吕晓丽[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《中国兽医学报》2008年第10期1118-1121,共4页Chinese Journal of Veterinary Science

基　　金：国家"十五"食品安全重大攻关专项基金资助项目(2001BA804A30-11)

摘　　要：根据已报道的猪细小病毒基因组序列,设计并合成了引物和探针,从猪细小病毒(PPV)感染的细胞中提取DNA,经PCR扩增,产物纯化后与pGEM-T-easy连接,转化大肠杆菌JM109,筛选后得到重组标准品质粒,对重组标准品质粒进行PCR和测序鉴定,表明目的片段已经成功克隆。将104～108拷贝反应的重组标准品质粒进行荧光定量PCR,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系。动力学曲线分析表明,在该反应体系和反应条件下,标准曲线的灵敏度为102拷贝。本方法的建立为猪细小病毒感染的早期诊断奠定了基础。According to genome sequences of porcine parvovirus （PPV） published in GenBank, A pair of primers and a TaqMan probe were designed. The expected fragment was amplified from DNA of PPV-infected PK-15 cells. The purified PCR product was connected with pGEM-T-easy vector and then transferred into JM109. The standard recombinant plasmid was gained from positive bacterium clone. The plasmid PCR and plasmid sequence mensuration showed that the expected fragment was successfully cloned. 10^1 -10^5 copies/uL. standard recombinant plasmid DNA specimen were amplified by real-time quantitative PCR,which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen. Analysis of the dynamic curve showed that under this condition of the reaction, the sensitive degree is 10^2 copies. The construction of realtime quantitative PCR provided the basis for the early diagnosis of PPV infection.

关 键 词：猪细小病毒 实时荧光定量PCR 标准曲线 

分 类 号：S852.65[农业科学—基础兽医学]