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为从临床样品中检测和分析SARS CoV病毒打基础 ,并为分析SARS CoV病毒的复制和转录等机理提供一种有效方法。以SARS冠状病毒TOR2株序列作为标准设计和制备一种覆盖SARS冠状病毒全基因组的寡聚核苷酸芯片 ,探针长度为 70nt,每相邻的探针序列重复 2 5nt,共660条。用该芯片分析了细胞培养的SARS CoV病毒总RNA、7个SARS CoV病毒的基因克隆片段。对RNA样品用随机引物进行反转录PCR获得cDNA。对DNA用随机引物扩增和dUTP cy3标记。结果用这种芯片杂交检测SARS CoV病毒RNA可见阳性信号呈全基因组分布 ,并且有多处连续的阳性信号点 ;用正常人的白细胞RNA为对照 ,杂交未出现明显阳性信号。检测 7个SARS CoV病毒基因克隆片段 ,在该片段相应的探针区段出现连续阳性信号点。这种方法可有效地检测和分析样品中SARS冠状病毒全基因组的信息。
In order to detect and analyze the foundation of SARS CoV virus from clinical samples and provide an effective method for the analysis of replication and transcription mechanism of SARS CoV virus. An oligonucleotide microarray covering the complete genome of SARS coronavirus was designed and prepared based on the sequence of SARS-CoV TOR2 strain. The length of the probe was 70nt, and the number of adjacent probe sequences was 2.55, a total of 660. The chip was used to analyze the total RNA of the cell culture SARS CoV virus and the gene clone fragments of seven SARS CoV viruses. Reverse transcription PCR was performed on RNA samples using random primers to obtain cDNA. DNA was amplified with random primers and labeled with dUTP cy3. Results The RNA of SARS CoV virus was detected by this chip hybridization. The positive signal showed the whole genome distribution, and there were many continuous positive signal points. With normal human leukocyte RNA as the control, no significant positive signal was found in the hybridization. Seven cloned fragments of the CoV viral gene of SARS were detected, and consecutive positive signal points appeared in the corresponding probe segment of the fragment. This method can effectively detect and analyze SARS coronavirus genome-wide information in samples.