HIV检测基因芯片的研制和应用评价

The preparation and application evaluation of DNA microarray genechip kit for HIV detection

目的研制用于HIV检测的基因芯片,并评价其在临床诊断和出入境检疫中的应用价值。方法针对HIV-1gags基因相对保守区域,设计检测探针,并设计内标和对照探针,制备HIV检测微阵列基因芯片;设计引物,以1:20引物比例不对称扩增HIV基因靶标,Cy3荧光标记扩增产物,作为杂交模板。Cy3荧光标记PCR扩增产物与微阵列探针杂交反应,结果经荧光扫描,并用分型软件分析判断阳性探针和样本HIV阴阳性。采用信号放大、梯度临界值技术,增加了检测的灵敏度和特异性。采用70例临床样本实样检测与DNA测序作对照,评价试剂盒的性能和应用价值。结果70例样本,采用本试剂盒与DNA测序并行检测,两者符合率为98.57%。结论本HIV检测基因芯片试剂盒具有高通量、操作简便、低成本、准确度好等优点,应用研究表明,在临床HIV诊断检测和出入境检测方面具有很高的应用价值。

Objective To develop a DNA microarray kit for HIV detection, and to evaluate the application value in clinical diagnosis and entry-exit inspection and quarantine. Methods The specific oligonucleotide probes for HIV-1 gags region and interior label or comparison probes were designed and spotted on glass slides using a microarray spotter. DNA sample were by amplified asymmetric PCR for HIV gags gene and the products of PCR were labeled with the fluorescent dye Cy3. The asymmetric PCR primer concentration ratio was 1 ： 20. Following hybridization between the Cy3-1abeled PCR products and the oligonucleotide probes of each microarray on the slides and wash, the amount of probe hybridizing to each DNA sample was measured with a microarray scanner and the image was analyzed with a computer software, which was used to identify the positive probes and to determine the HIV on each sample. Results There was a good concordance between the genotyping results by oligonucleotide microarray and those of SBT. 70 samples were tested and the coincidence rate was 98.57%. Conclusion The procedure described here has the potential of providing a rapid, simple, economical and accurate way for HIV detection, which has high application value in clinical diagnosis and entry-exit inspection and quarantine.