DNA芯片检测乙型肝炎病毒基因多态性

目的　建立DNA芯片检测乙型肝炎病毒 (hepatitisBvirus,HBV)基因多态性的研究方法并对实验条件进行优化。方法　设计多条寡核苷酸探针 ,在硅烷化芯片的特定位置上 ,用点样仪将探针固定 ,并与PCR扩增的HBV基因相应区段杂交 ,杂交结果影印至硝酸纤维素膜 ,经BCIP NBT避光显色 ,用放大镜观察杂交信号呈暗紫色圆点 ,根据特定位置上杂交信号的有无和与之相应的探针序列来判定基因突变的类型。结果　通过 1次杂交反应可检测HBV前C C区 (nt 1896 1814 )、BCP区 (nt1762 1764)和P区 (nt 52 8 552 )等多个位点的变异 ,与测序分析结果完全一致 ,具有较好的检测灵敏度和重复性。结论　DNA芯片检测HBV基因常见突变位点多态性 ,操作简便易行 ,技术要求不高 ,具有临床推广应用价值 ,而且可以方便地通过向寡核苷酸探针阵列中添加相应探针 ,扩大基因芯片的检测应用范围 ,为临床检测提供了新的方法

Detection of polymorphism of hepatitis B virus gene by DNA microarray

Objective To detect HBV gene polymorphism by DNA microarray and to identify influential factors of the assay. Methods HBV PCR products were hybridized with oligonucleotide probes, which were pre-stablized on the chip by Cartesian Pixsys 7500. The hybridized result s were colorized by BCIP/NBT on nitrocellulose filter(NC). According to the hybr idization signal (dark purple dot) and the corresponding probe sequence, mutatio n type was determined. Results Multiple mutation sites of HBV including precore/core region(nt 1896/ 1814 ), BCP region(nt 1762/1764 )and P region(nt 528/552)were identified by single hybridization reaction, an d the results were identical with sequencing analysis. The assay was sensitive a nd specific. Conclusion This assay was simple and easy to carry out, and the detective range of DNA microarray can be further extended by adding oligonucleotide probes to it without significantly increasing complexity or cost.