The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone h...The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.展开更多
该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;...该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green Ⅰ荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段属于P1,所建立的SYBR Green Ⅰ荧光定量PCR检测P1的反应在101-108拷贝/μL之间具有良好的线性关系,反应的检出下限为10拷贝/μL,而对猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒等的检测为阴性,表明该方法敏感、特异。成功建立了SYBR Green Ⅰ荧光定量PCR检测P1载量的方法,为P1致病机制和机体免疫保护机制的研究提供了技术平台。展开更多
基金supported by the State Key Basic Research Project(973 project)of China(Grant No.2007CB116308)Planned Projects for Postdoctoral Research Funds of Jiangsu Province,China(Grant No.5910602)Postdoctoral Funds of Jiangsu Academy of Agricultural Sciences,China(Grant No.6510501)
文摘The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1.
文摘该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green Ⅰ荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段属于P1,所建立的SYBR Green Ⅰ荧光定量PCR检测P1的反应在101-108拷贝/μL之间具有良好的线性关系,反应的检出下限为10拷贝/μL,而对猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒等的检测为阴性,表明该方法敏感、特异。成功建立了SYBR Green Ⅰ荧光定量PCR检测P1载量的方法,为P1致病机制和机体免疫保护机制的研究提供了技术平台。