乙型肝炎、丁型肝炎病毒联合诊断芯片制备的初步研究

Microarrays for detecting HBV and HDV simultaneously

目的 制备联合检测乙型肝炎病毒 (HBV)、丁型肝炎病毒 (HDV)基因芯片并进行杂交验证。方法 利用PrimerPremier5 0分别针对HBV、HDV基因保守区域设计多对PCR引物 ,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鉴定。用PixSys 5 5 0 0芯片打印仪将PCR产物打印在氨基修饰的玻片上制备成检测芯片。样品荧光标记采用限制性显示 (RD)技术 ,标记后进行杂交验证分析。结果 序列分析表明 ,运用PCR技术得到的多个基因片段均属于HBV、HDV特异基因。杂交结果显示 ,敏感性、特异性、重复性等指标均佳。结论 利用PCR扩增产物作为探针制备HBV、HDV联合诊断芯片是一种快速、简便的实用方法 ,有着广阔的应用前景 ;利用RD技术标记样品可提高多种肝炎病毒混合检测的敏感性。

Objective To prepare the microarrays for joint detection of HBV and HDV. Methods The specific primers of PCR were designed with Primer Premier 5.0 program according to the conserved regions 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 microarray was obtained by spotting PCR products onto the surface of glass slides by robotics. Restriction display PCR (RD-PCR) was used to label the samples. Results After the sequences were aligned, we found that the products of PCR amplification were the specific gene fragments of HBV and HDV. The hybridized signals on gene chips indicated that the specificity and sensitivity of DNA microarray for joint detecting the HBV and HDV were satisfactory. Conclusion Using PCR amplification products to construct gene chips is a quick, simple and effective method for clinical diagnosis of HBV and HDV. Further application of restriction display PCR technique in labeling the sample may expedite and raise the sensitivity in multi-virus detection by microarray technology.