摘要
目的建立能对虫媒病毒进行属水平筛查及种水平鉴定的基因芯片技术,为虫媒病毒提供一种高通量的检测与鉴定技术。方法从GenBank获得13种虫媒病毒的全部基因组序列,利用Clustal X和BLAST等比对软件,设计针对这些虫媒病毒所在的3个属的通用寡核苷酸探针,用于病毒的属水平筛查。然后设计针对每种病毒的特异性寡核苷酸探针,用于每种病毒的检测鉴定。寡核苷酸探针长度均为70mer,将设计好的探针与GenBank上所有病毒基因组序列进行比对,验证其种属特异性,然后进行探针合成和基因芯片制备。所有病毒在BSL-3和BSL-2实验室进行培养,待检病毒核酸通过锚定随机PCR进行扩增,经荧光染料标记后与基因芯片杂交,利用激光共聚焦扫描仪扫描后进行结果分析。结果所检测的13种虫媒病毒均可获得种特异性及所在属的属特异性杂交信号,非特异性杂交信号不明显。分别用两种不同的病毒混合后,进行模拟混合病毒感染,也得到了相应的特异杂交信号。结论初步建立了13种虫媒病毒的基因芯片检测技术,可用于这些病毒的属水平筛查及种水平鉴定,并且可以进行混合感染标本的检测,为虫媒病毒的检测与鉴定提供了一种新的手段。
Objective Arbovirus could induce severe symptoms in human such as febrility, erythra, arthralgia, hemorrhagic fever and shock. Biochip is a new technique which has been developed since 1990＇s. The significant advantages of biochip are high throughput, high parallel and high autornation. 13 species of arboviruses were selected in present study to establish DNA microarray for screening and identifying those viruses both in genus and species levels. Method all known genome sequences of the above arboviruses were downloaded from GenBank Then genera-specific and species-specific oligonucleotides were designed by Clustal X and BLAST. Oligos of Flavivirus, Alphavirus and Orthounyavirus were designed for genus-spesific screening and species-spesific oligos for each species were designed for specific identificatior, All oligo-probes were 70mer in length. The designed species and genus-specific probes were validated firstly on Internet for their specification by BLAST. And then the optimized oligos were synthesized and spotted on chips. Viruses were cultivated in BSL-2 and BSL-3 labs. Anchoring random PCR was performed to amplify viruses RNA, The PCR products were labeled by fluorochrome incorporatior, Hybridization and washing conditions were optimized. The hybridization results were analyzed by ScanArray software. Results Specific hybridization signals were obtained in 13 species- and 3 corresponding genera levels, and non-specific signals were obsolete. Specific signals were also observed in mixed specimens. Conclusion A DNA microarray technique has been constructed for the detection of 13 species of arboviruses. It could be used for screening and identifying those viruses both in genus and species levels. It could also be used to detect mixed specimer, This technique provided a novel method for the detection of arboviruses.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第8期832-835,共4页
Medical Journal of Chinese People's Liberation Army
关键词
寡核苷酸序列分析
虫媒病毒
检测
oligonucleotide array sequence analysis, arboviruscs
detection
