【摘要】 通过分子克隆技术获得马疱疹病毒1型(Equineherpesvirus1,EHV1)、马动脉炎病毒(Equinearteritisvirus,EAV)、马流感病毒(Equineinfluenzavirus,EIV)、马传染性贫血病毒(Equineinfectiousanaemiavirus,EIAV)和东部马脑脊髓炎病毒(East-ernequineencephalomyelitisvirus,EEEV)等5种病毒各一段高度保守的特异性基因片段,用芯片点样仪逐点分配到处理过的玻片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测和计算机软件分析。结果显示,制备的基因芯片可同时检测和鉴别上述5种病毒,可检测到阳性杂交信号的最高稀释度为10-6的病毒液,约25个病毒DNA拷贝,但其它病毒材料未见红色荧光信号,证明了本方法的特异性。在进口马的隔离检疫期间,采集马鼻肺炎、马动脉炎中和抗体阳性但病毒分离阴性马匹的白细胞悬液,分别在EHV1和EAV位点处可检测到阳性杂交信号。证明基因芯片技术不但快速、准确和敏感,而且可同时进行多种病毒的检测。更多还原

【Abstract】 The highly conserved DNAs of Equine herpesvirus 1(EHV1), Equine arteritis virus(EAV), Equine influenza virus(EIV), Equine infectious anaemia virus (EIAV) and Eastern equine encephalomyelitis virus (EEEV) were acquired by molecular cloning, and then spotted on the treated glass slides as the diagnostic gene-chip. And the cDNAs reverse-transcripted from RNAs of samples were labeled with Cy5 fluorescence as probes. Following specific hybridization of deposited gene chip and labeled probes, fluorescence signals were scanned by laser scanner and the obtained image was analyzed by QiamtArray software with the digital computer. The results showed that the prepared gene chip could detect and distinguish the five equine viruses. And its sensitivity was about 25 copies of viral genomes. The hybridization specificity was confirmed by the presence of red fluorescence signals on the corresponding sites with samples from the five relevant viruses in horses and by the absence of positive signals with the specimens from irrelevant viruses in other animals. Peripheral blood leucocyte (PBL) from some seropositive horses of post-arrival quarantine was negative by virus isolation but positive for EHV1 and EAV by gene chip. The evidence suggests that gene chip, which is quick, specific, sensitive, and reliable, can provide a practical alternative to screen and quarantine a large number of samples within a very short period of time.更多还原