纳米金比色芯片同步目视化检测HBV和HCV

Detection-visualized oligonucleotide array for HBV and HCV coinfection with Au nanoparticle probes

目的建立一种简单、快速的比色芯片可同时目视化检测 HBV DNA 和 HCV RNA。方法利用多重 PCR 方法扩增 HBsAg 阳性合并抗 HCV 阳性的患者血清中提取的 HBV DNA 和 HCVcDNA。制备 Au 纳米颗粒基因探针,检测探针与固定在尼龙膜上的捕捉探针构成双探针,用斑点杂交法检测 HBV DNA、HCV cDNA 的多重 PCR 产物,加入银离子(Ag^+)-对苯二酚液染色观察结果。结果多重 PCR 可同时扩增 HBV DNA 和 HCV cDNA,出现预期的431bp 和323bp 特异性条带。纳米金比色芯片可以同步目视化检出乙型、丙型肝炎合并感染患者血清中 HBV DNA 和 HCV RNA 的 PCR 产物。结论目视化 HBV、HCV 纳米金比色芯片诊断方法操作简单、特异性好、成本低廉、结果判定指标客观,此类基因芯片在病毒基因检测领域将会有广泛用途。

Objective To establish a simple and rapid visual method based on nanogold supported probes for detecting HBV and HCV simultaneously on DNA microarray. Methods Multi-primer PCR identifying HBV and HCV simultaneously in serum for patients with HBV and HCV coinfection was established. With the aid of Au nanoparticle-supported mercapto-modified oligonucleotide serving as detection probe, and oligonucleotide immobilized on nylon membrane surface acting as capturing probe, HBV and HCV PCR products were detected visually by sandwich hybridization based on highly sensitive aggregation and silver staining. Results HBV DNA and HCV RNA can be specifically amplified simultaneously in one system by multi-primer PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. HBV and HCV PCR products could be detected visually with this oligonucleotide array. Conclusions The established oligonucleotide array for the detection of HBV and HCV coinfection is convenient and efficient with high specificity and sensitivity. It provides a nationale for the measurement of other viruses coinfection.