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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 prepare a combined DNA microarray for hepatitis B and hepatitis B virus (HBV) gene and verify its hybridization. Methods Primer 5.0 software was used to design multiple pairs of PCR primers for conserved regions of hepatitis B and D viruses respectively. The PCR products were purified and cloned into pMD18 T vector. The positive clones were extracted and sequenced. Identification. The PCR product was spotted on a glass slide using a chip spotter to prepare a gene chip. Sample markers using restrictive display technology, the sample is marked after hybridization. Results Using PCR technology, a number of hepatitis B and hepatitis B virus-specific gene fragments were obtained. Sequence analysis showed that the amplified fragments belong to HBV, HDV specific gene. Hybridization results show that the sample and hepatitis B, hepatitis D diagnostic gene chip hybridization sensitivity and specificity are good. Conclusion It is a quick and easy practical method to prepare B type and D type diagnostic gene chips by PCR amplification products, which has a broad application prospect. In addition, the use of restrictive display technology to label samples may provide a foundation for further testing of a variety of hepatitis viruses.