人类致病病毒属水平基因筛查芯片的制备及其在黄病毒属检测中的初步应用

Preparation of DNA microarray for screening human pathogenic viruses and its preliminary application to detection of flavivirus

目的：制备人类致病病毒属水平高通量筛查用基因芯片，并通过检测黄病毒属病毒初步验证其筛查效果。方法：利用Clustal W和BLAST软件设计针对人类致病病毒的属特异性寡核苷酸探针，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优化。分别用特异或随机PCR方法从病毒感染的细胞培养上清中扩增病毒核酸，在扩增过程中掺入aa-dUTP并进一步偶联cy5或cy3荧光素进行标记，随后与基因芯片进行杂交，并通过荧光扫描仪对杂交结果进行分析，然后分析检测的特异性和敏感性验证基因芯片的筛查效果。结果：共设计了针对人类医学病毒的1090条寡核苷酸探针及其他阳性、阴性对照探针，其中黄病毒属共46条探针，Tm值为77，67～83．53℃，通过随机或特异PCR扩增8株黄病毒属病毒核酸，在42℃、过夜杂交的条件下，通过基因芯片方法均获得了黄病毒属特异性杂交信号，与其他属病毒之间没有非特异性交叉反应。结论：所制备的基因芯片可用于黄病毒属病毒的快速筛查，本研究为进一步建立大规模病毒性病原体的高通量筛查与鉴定技术平台提供了实验依据。

Objective：To establish a high throughput DNA microarray method for rapidly screening human viral pathogens, and to verify its effectiveness by detecting Flavivirus. Methods： ClustalW and BLAST software were employed to design the genus-specific oligonucleotides probes for all known human pathogenic viruses. The concentration of probes, hybridization temperature and duration, formulation of hybridization solution and washing conditions were optimized. Specific or random-PCR was used to amplify virus-targeting nucleic acids from supernatant of virus-infected tissue culture cells. Aminoallyl-dUTP was incorporated into the PCR products, and PCR products were further labeled with Cy5 or Cy3. After microarray hybridization, all arrays were imaged and hybridization signals were analyzed. The effectiveness of DNA microarray was validated by measuring the specificity and sensitivity of the method. Results：Totally 1090 oligonucleotides were designed for all known human pathogenic viruses, and other 24 oligonucleotides were selected as positive and negative controls. Of them, 46 were designed as probes for flavivirus and the Tm value were between 77.67 -83.53℃. After hy- bridization at 42℃ overnight, 8 strains of viruses had detectable flavivirus-specific hybridization signals. No non-specific hybridization signals were observed. Conclusion：DNA microarray strategy could be employed for rapid screening of flavivirus at genus level, it would be feasible to further establish DNA microarray platform for high throughput screening and identification of unknown viral pathogens.