3种常见呼吸道病毒检测基因芯片的制备

A gene chip to detect three types of common respiratory viruses

目的建立一种可同时检测鼻病毒、呼吸道合胞病毒以及腺病毒的基因芯片方法。方法根据GenBank已发表的鼻病毒、呼吸道合胞病毒和腺病毒的基因序列,应用生物学软件设计相应的引物和寡核苷酸探针,构建检测芯片;使用巢式PCR扩增标记目的片段,将其与检测基因芯片进行杂交和扫描分析;通过特异性、灵敏性和重复性试验评估基因芯片方法。结果设计的全部探针均能与相应的标记样品特异性杂交,其中探针1a和1b可特异检测鼻病毒;探针2a、2b和2c可特异检测呼吸道合胞病毒,探针3a和3b可特异检测腺病毒。基因芯片法检测3种病毒的最低拷贝数分别是2.59×102个/μl、2.21×101个/μl和2.05×102个/μl。结论构建的基因芯片可特异性检测样品中低滴度的鼻病毒、呼吸道合胞病毒以及腺病毒,有望为临床提供确诊依据。

Objective To establish a quick and sensitive method to simultaneously detect rhinovirus, respiratory syncy-tial virus, and adenovirus using gene chips. Methods Oligonucleotide probes and primers were designed with bioinfor-matics software based on sequences of rhinovirus, respiratory syncytial virus, and adenovirus in GenBank and oligonucle otide microarrays were prepared. Amplified by RT-nPCR and labeled, the fragments were hybridized with probes on the gene chip and then scanned and analyzed to evaluate the use of oligonucleotide microarrays in terms of specificity, sensitiv-ity, and reproducibility. Results Probes were all hybridized with correspondingly labeled samples and had strong posi- tive signals： Probes la and lb specifically detected rhinovirus, probes 2a, 2b, and 2c specifically detected respiratory syn-cytial virus, and probes 3a and3b specifically detected adenovirus. The minimum number of copies a sample should have was 2.59 × 102 ind/μl, 2.21 × 10^1 ind/μ1, and 2.05 × 10^2 ind/μl, respectively. Conclusion The prepared gene chips specifically detected very low titers of rhinovirus, respiratory syncytial virus, and adenovirus in samples. These chips have the potential to definitively diagnose these viruses in clinical settings.