HPV病毒检测分型芯片的研制和优化

Development and Optimization of Oligonucleotide Microarray for Detection and Sub-typing of Human Papillomavirus

研制和优化寡核苷酸芯片以初步实现对多种常见HPV（Human papillomavirus）病毒的分型检测．应用生物学软件对四型常见HPV病毒（6、11、16、18型）的全基因组序列进行分析，设计具有型特异性、熔解温度（Tm）相近的-60mer寡核苷酸探针，对玻片片基进行优化处理后，点样制备成寡核苷酸基因芯片．将含HPV全长基因序列的质粒作为阳性标准品，利用梯度限制性荧光标记技术对其进行荧光标记，标记好的样品与芯片杂交．结果显示HPV样品与相应的型特异性探针杂交有明显的荧光信号，而与阴性对照探针和空白对照探针没有杂交信号．通过对芯片片基处理和样品荧光标记方法的优化，可以提高芯片检测的杂交特异性和荧光信号强度．

An oligonucleotide microarray assay for detection and sub-typing of human papillomavirus （HPV） was developed and optimized. Biological softwares Arraydesigner 2.0 and BLAST program were applied to analyze the whole genome of four different HPV types（6, 11, 16 and 18） to design - 60 mer oligonucleotide probes with high specificity and similar melting temperature （Tm）. The cultured HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display PCR（RD-PCR）. The labeled samples were hybridized with the oligo microarray. HPV DNA samples were hybridized specifically with multiple spots correspondingly to show positive signals and the corresponding HPV subtypes were recognized as well, while no signals were detected of all the negative and blank controls. With optimization of glass slide treatment and fluorescence labeling method, the hybrid specificity and fluorescence signal intensity of oligo microarray were heightened.