多种病毒集合检测芯片的研制及其在临床血清标本检测中的初步应用

Research on microarray for simultaneous detection of multiplex viruses and primary application on detection of clinical serum specimens

目的研制黄热病、西尼罗热、登革热、基孔肯雅热、埃博拉出血热、马尔堡出血热、拉沙热等多种病毒性传染病集合检测基因芯片,并建立一种适用于直接检测核酸含量较低临床血清标本的新型芯片靶基因扩增标记方法。方法设计并筛选出上述病毒70mer寡核苷酸特异探针各20条,打印于同一基因芯片上;以phi29 DNA聚合酶结合带标签序列的随机引物进行临床标本中病毒全基因组扩增,再以Cy3荧光染料标记的标签序列引物进行PCR随机扩增标记,标记产物用多种病毒芯片进行杂交检测。结果检测1～4型登革热、基孔肯雅热病例临床血清标本,结果显示该方法准确、特异、敏感;检测西尼罗热、黄热病、埃博拉出血热、马尔堡出血热、拉沙热等病毒核酸的模拟血清标本,同样得到特异的阳性杂交信号,与预期结果一致。结论本研究建立的多种病毒集合检测基因芯片及其靶基因扩增标记方法,可直接应用于血清标本中上述病毒核酸的检测。

Objective To develop a microarray for simultaneous detection of multiplex viruses,including Yellow fever,West Nile fever,Dengue fever,Chikungunya fever,Ebola hemorrhagic fever,Marburg hemorrhagic fever,Lassafever,and set up a new target gene amplification method for direct detection of clinical specimens with lower nucleic acids.Methods 20 specific 70mer oligonucleotide probes for each virus were designed and screened,that printed on the same chip;Whole genome amplification of viruses were performed by using phi29 DNA polymerase and a random primer containing tag sequence,then amplified and labeled by random PCR with a Cy3-conjugated tag sequence primer.Finally,hybridization and detection were conducted with the chip.Results The detecting method for viruses of dengue fever（type 1-4） and chikungunya fever in clinical serum specimens were accurate,specific and sensitive;Meanwhile,The detection results for viral nucleic acids of West Nile fever,Yellow fever,Ebola hemorrhagic fever,Marburg hemorrhagic fever,Lassa fever in simulated clinical serum specimens,also showed specific positive hybridization signal with the expected results.Conclusions The microarray established in this study for simultaneous detection of multiplex viruses and the new target gene amplification method,can be directly applied to the detection of viral nucleic acids in serum specimens.