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目的 设计并应用60 mer寡核苷酸基因芯片实现对SARS冠状病毒的检测。方法 根据寡核苷酸基因芯片的设计原则设计出3O条60mer的寡核苷酸片段,覆盖SARS冠状病毒(以TOR2株为参考序列,GENEBANK索取号:AY274119)全基因组,点样制备成12×12的寡核苷酸基因芯片,将临床诊断SARS患者痰液标本抽提病毒RNA,采用限制性显示技术进行标记,将标记好的样品与芯片杂交,洗脱,扫描。初步探索寡核苷酸芯片诊断SARS冠状病毒的敏感性及特异性,并对芯片做进一步的优化。结果 来自SARS患者样品与芯片上的多个SARS探针杂交,出现了明显的荧光信号;阴性和空白对照探针上没有杂交信号,而对照样品仅与一个SARS探针有杂交。结论 采用60 mer寡核苷酸基因芯片可以从基因水平实现对SARS冠状病毒多段序列的平行检测,对于提高检出率有一定意义,此外,尚可用于监测在发病过程的各个阶段中病毒基因的活动状况。
Objective To design and use 60 mer oligonucleotide microarray to detect SARS coronavirus. Methods According to the design principles of oligonucleotide microarray, 30 oligonucleotide fragments of 60mer were designed to cover the whole genome of SARS coronavirus (TOR2 strain as reference sequence, GENEBANK accession number: AY274119) 12 oligonucleotide microarray gene chip, sputum samples of clinically diagnosed patients with SARS extracted viral RNA, using restrictive display technology for labeling, the labeled sample and the chip hybridization, elution, scanning. To explore the sensitivity and specificity of oligonucleotide microarrays in the diagnosis of SARS coronavirus and to further optimize the chip. RESULTS: Samples from SARS patients hybridized to multiple SARS probes on the chip with a clear fluorescent signal; no hybridization signal was observed on the negative and blank control probes, whereas the control sample hybridized to only one SARS probe. Conclusion The 60 mer oligonucleotide microarray allows parallel detection of multiple sequences of SARS-CoV at the genetic level, which is of significance in improving the detection rate. In addition, it can be used to monitor the expression of viral genes in various stages of the pathogenesis Activity status