人兽共患病病毒基因芯片检测敏感性的测定

Determination of gene-chip sensitivity on detecting zoonotic viruses

目的测定人兽共患病病毒基因芯片检测的敏感性,为建立高通量的人兽共患病病毒检测基因芯片提供依据。方法以黄病毒属的日本脑炎病毒(JBEV)作为检测敏感性的模型,以随机PCR扩增法扩增病毒基因组,然后将扩增产物标记荧光染料后与基因芯片进行杂交,以两种方式测定芯片的敏感性,即最低检出核酸量和最低检出病毒TCID50量。最后,与特异PCR敏感性比较,评估本芯片的检测敏感性。结果人兽共患病病毒基因芯片至少可以检测出300ng的随机PCR产物核酸量。对病毒的检测敏感性可以达到10倍稀释的TCID50病毒量(即每200μl含有105TCID50的病毒量),与特异PCR检测敏感性相当。结论人兽共患病病毒芯片技术达到了较高的检测敏感性;建立高通量的人兽共患病病毒检测基因芯片技术具有可行性与实用性。

Objective To determine the gene-chip sensitivity on detecting zoonotic viruses, and found a basis for large scale screening and identifying zoonotic viruses by gene-chip technique. Methods Japanese encephalitis virus （JEV） of genus Flavivirus was chosen as the model to determine the sensitivity of the gene-chip. The virus genome was amplified by random PCR, and the PCR products were then labeled with fluorescence dye and hybridized with zoonotic viruses gene-chip. Two methods were employed to determine the sensitivity of the gene-chip. One was to determine the minimal quantity of random PCR products, and the other was to detemaine the minimal TCI^0. Finally, the sensitivity of zoonotic virus gene-chip was also compared with that of specific RT-PCR Results The minimal quantity of ran- dom PCR products＇detected by the zoonotie virus gene-chip was 300ng, and the detective limitation of virus titer was 10 folds of TCID50 dilution, i.e.10^5 TCID50/200μl. The sensitivity of the gene-chip was equivalent to that of specific RT PCR. Conclusion The zoonotic virus gene-chip achieves a high sensitivity, indicating that to establish a large scale screening for identifying zoonitic viruses by gene-chip-based detection is feasible and practical.