乙型、丁型肝炎病毒诊断基因芯片制备的初步实验研究

Microarrays for detecting HBV and HDV

目的 制备联合检测乙型、丁型肝炎病毒基因芯片并进行杂交验证。方法 利用Primer 5 .0软件分别针对乙型、丁型肝炎病毒基因保守区域设计多对PCR引物 ,纯化PCR扩增产物 ,扩增后的产物克隆至 pMD18 T载体 ,提取阳性克隆质粒进行测序分析及鉴定。用芯片点样仪将PCR产物点到玻片上制备成基因芯片。样品标记采用限制性显示技术 ,样品标记后进行杂交。结果 运用PCR技术 ,得到多个乙型、丁型肝炎病毒特异性基因片段。序列分析表明 ,所扩增的片段均属于HBV、HDV特异基因。杂交结果显示 ,样品和乙型、丁型肝炎诊断基因芯片杂交的敏感性和特异性均佳。结论 利用PCR扩增产物制备乙型、丁型诊断基因芯片是一种快速、简便的实用方法 ,有着广阔的应用前景。另外 ,利用限制性显示技术标记样品可为进一步更多种肝炎病毒的混和检测奠定基础。

Objective To study the Microarrays for HBV and HDV detection.Methods The specific primers of PCR were designed with the Primer 5.0 program according to th conserved region of HBV and HDV. The PCR fragments were purified and cloned into the pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarrays were prepared by spotting the PCR products to the surface of glass slides by the robotics. Restriction Display PCR (RD-PCR) was used to label the samples. Results Sequences were aligned, and the results showed that the products of PCR amplification were specific gene fragments of HBV, HDV. From the hybridizing signals on Gene chip, the specificity and sensitivity in detecting the HBV and HDV were satisfactory. Concision Using PCR amplification products to construct the gene chip for clinical diagnosis of HBV and HDV is a quick, simple and effective method. Further application of Restriction Display PCR (RD-PCR) technique in labeling the samples can speed up the muti-viral detection by microarray technology.