非洲马瘟病毒、西尼罗病毒和马冠状病毒的基因芯片检测技术研究

Development of Gene Chip Method for Detecting AHSV,WNV and ECV

为探索建立马病病毒基因芯片检测方法,采用人工拼接的方式拼接了非洲马瘟病毒(ASHV)核酸序列,通过分子克隆技术获得西尼罗病毒(WNV)和马冠状病毒(ECV)的特异基因片段。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。以拼接、克隆的核酸序列为模板通过多重不对称RT-PCR进行特异性扩增和荧光标记后滴加到芯片上进行杂交,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3种病毒,ECV质粒样品、WNV质粒样品检测灵敏度为102拷贝;AHSV质粒样品检测灵敏度为104拷贝。其他病毒材料未出现荧光信号,验证了本方法的特异性。证明基因芯片技术可快速、准确和灵敏地同时进行多种病毒的检测。

In order to establish a gene-chip detection method of equine virus, the highly conserved DNAs of African horse sickness virus （AHSV） was obtained by artificial splicing, West Nile virus（WNV） and equine corona virus（ECV） were ac- quired by molecular cloning, and then spotted on the treated glass slides as the diagnostic gene-chip. And the cDNAs amplified by multiple asymmetric PCR with the template of splicing and cloned nucleic acid sequence were labeled with 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 Qiamt Array software with the digital computer. The results showed that the prepared gene chip could detect and distinguish the three equine viruses. And its sensitivity was about 104 copies of AHSV and l0s of ECV and WNV. The hybridization specificity was confirmed by the presence of fluorescence signals on the corresponding sites with samples from the three relevant viruses DNA template and by the absence of positive signals with the specimens from irrelevant viruses was negative by gene chip. The evidence suggested that gene chip, which was quick, specific, sensitive, and reliable, could provide a practical alternative to screen and quarantine a large number of samples within a very short period of time.