基因芯片技术检测10种烈性RNA病毒  

The detection of 10 violent RNA viruses by microarray technology

作　　者：杨银辉[1] 杨瑞馥[1] 常国辉[1] 司炳银[1] 翟俊辉[1] 吕富双[1] 周东生[1] 韩伟国[1] 祝庆余[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《解放军医学杂志》2006年第3期203-206,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家科技攻关计划资助课题(2003BA712A01)

摘　　要：目的利用基因芯片技术检测包括披膜病毒科甲病毒属、黄病毒科黄病毒属、布尼亚病毒科汉坦病毒属及SARS病毒等10种烈性RNA病毒。方法设计了甲病毒属的属保守区通用引物和黄病毒属的通用引物,与布尼亚病毒及SARS病毒的引物一起用于扩增靶核酸。另外,在通用引物所能扩增的区域内设计每种病毒的特异引物,利用该引物通过PCR反应制备cDNA克隆探针,在判定其特异性后,制备氨基化探针。对探针浓度、杂交温度、杂交时间、不同杂交液及杂交后芯片的洗涤方法进行了优化。结果核酸探针浓度为0·3μg/μl时,可获得杂交信号;在杂交液终浓度含20%甲酰胺、杂交60℃、1·5h的条件下,可分别获得以上10种病毒的特异杂交信号。利用多重PCR扩增靶序列时,可获得2种或4种混合病原体的特异性杂交信号。结论利用基因芯片技术检测病毒性病原体是可行的,关键在于设计合适的引物及探针序列。Objective To detect 10 RNA viruses including Alphavirus in Togoviridae, Flavivirus in Flaviridae, Hantavirus and Nairovirus in Bunyavirudae and SARS-CoV in Coronavirudae by using genechip technique. Methods The universal PCR primers of Alphavirus and Flavivirus and the PCR primers specific for HFRSV in Hantavirus and XJHFV in Nairovirus were designed by DNAStar sftware. PCR primers specific for SARS-CoV were adopted from WHO website. In addition, all the PCR primers specific for each virus were designed inside the regions of universal primers. These specific primers were utilized for amplification of cDNA probes. The concentration of probes, the hybridization temperature and duration, the formulation of hybridization .solution and the washing conditions were optimized. Results The specific hybridization signals could be obtained when the concentration of probes was 0. 3ug/ul. Good hybridization signals could be obtained for all the 10 RNA viruses when the hybridization solution contained 20% formamide, and the hybridization reaction was conducted at 60℃ for 1.5 hours. Two or four pathogens could be detected simultaneously when the target nuclear acids were amplified by multiplex PCR Conclusion The results showed that the virus pathogens could be detected by genechip technique, and the key step was to design suitable primers and probes.

关 键 词：寡核苷酸序列分析 RNA 病毒 检测 

分 类 号：R373-33[医药卫生—病原生物学] R446.52[医药卫生—基础医学]