猪伪狂犬病病毒检测基因芯片的构建

Construction of gene chip for detection of porcine pseudorabies virus

为了研制猪伪狂犬病病毒（pseudorabies virus,PRV）诊断DNA芯片,选取PRV gE基因设计引物与探针以构建DNA芯片,同时对基因芯片的探针质量浓度、Poly（d T）的添加、杂交温度、杂交时间进行筛选。结果显示：寡核苷酸探针的浓度在1-30μmol/L之间对杂交信号的影响不大;寡核苷酸探针的氨基化端加上Poly（d T）,49.5℃杂交1 h可以获得较好的杂交信号;敏感性试验结果显示,敏感性可达1×10^-6ng级,与普通PCR相比高10倍,该芯片具有特异性高、灵敏度高等优点;应用猪伪狂犬病毒检测基因芯片检测20份临床病料,与PCR结果符合率达100%。

This study was to develop a DNA chip for diagnosis of DNA-Pseudorabies virus. The g E gene of DNA-Pseudorabies virus was chosen,and the primers and probes were designed to construct DNA microarray. Screening of the mass concentration of the probe on the gene chip and the addition of Poly（ d T） carried out; and the hybridization temperature and the hybridization time were determined. The results showed that the concentration of the oligonucleotide probe between 1 and 30 μmol/L had little effect on the hybridization signal. The amination of the oligonucleotide probe with Poly（ d T） and 49. 5 ℃ hybridization at 1 h were more favorable for obtaining the hybridization signal.The chip had the advantages of high specificity,high sensitivity and reusability. The results of application of PRV DNA microarray to detection of 20 clinical samples were 100% in line with those of the PCR.