乙型脑炎病毒可视化分型基因芯片的制备

目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。

Preparation of Visual Microarrays to Genotype Japanese Encephalitis Virus

Objective： To prepare visual gene chips to genotype Japanese encephalitis virus（JEV）. Methods： According to genomic sequence of JEV, the primers and probes were designed to genotype JEV- Ⅰ and JEV-Ⅲ by applied biological software. The targeting fragments were amplified by PCR amplification with biotin labeling primers, subsequently, they were hybridized with probes fixing on the glass slide. Then, streptavidin modified nanogold was added to hybridize with the biotin, finally sliver enhancement was used to visualize hybridization reactions above. Specificity, sensitivity and repeatability were tested to verify this method. Results： The microarrays gave strong positive binding signal to labeled targeting fragment of JEV- Ⅰ or JEV-Ⅲ. The No.2 probe was able to detect JEV, and No.3 and 4 probes could genotype JEV- Ⅰ and JEV-Ⅲ respectively. Sensitivity of the gene chips was 10^5 copies/mL of JEV plasmids. The chips specifically reacted to JEV, but not five control virus including porcine reproductive and respiratory syndrome virus. The gene chips had stable repeatability. Conclusion： The prepared visual microarray method has high specificity, sensitivity and repeatability, suggesting it can be used to rapid genotype JEV in a high throughput way .