猪瘟病毒和猪细小病毒检测基因芯片的构建

本试验克隆猪瘟病毒(Classical swine fever virus,CSFV)和猪细小病毒(Porcine parvovirus,PV)各自病毒基因保守序列,提取质粒中的模板、扩增纯化作为探针,应用微量点样技术将探针固定在硝酸纤维素膜上,制备诊断基因芯片;同时对制备的基因芯片进行有效性监控和特异性、重复性的质量控制;然后提取样品核酸,PCR扩增并用生物素标记,并将所获得的扩增产物与诊断基因芯片进行特异性的逆向点杂交,最后通过芯片扫描来实现对病原的高效检测和分析判断。试验结果表明病料中抽提的核酸与芯片杂交的信号为阳性且较明显,说明制备的猪瘟病毒和猪细小病毒检测基因芯片有效性监控正常且两种病原的特异性和重复性均良好。

Construction and Application of Gene Chip for Classical Swine Fever Virus and Porcine Parvovirus

In this experiment, the conservative gene sequences of swine fever virus and porcine parvovirus were cloned and plasmid templates were extracted, amplified and purified as probes. The probes were fixed onto nitrocellulose membrane by micro-printing technology to construct the gene chip. And the efficiency, specificity and repetitive quality of gene chips were validated. Then the DNA were extracted from samples, amplified by PCR and marked with biotin. The specific reverse hybridization was conducted between the amplified products and diagnostic gene chips. Finally, the effective analysis and the judgement of the two pig pathogens were achieved by scanning the chips. The results showed that the hybridization signal between nucleic acid and chip was positive and obvious, the efficiency was normal and both the specificity and repetition were good against the two viruses.