用SARS冠状病毒全基因组芯片杂交方法分析SARS-CoV

Analysis of SARS Coronavirus by Oligonucleotide(70mer) Microarray Hybridization

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。

A new analyzing method for SARS coronavirus(SARS-CoV) and its early clinical diagnosis were presented. A whole viral genome chip for SARS-CoV was designed based on the genomic sequence of SARS-CoV TOR2 obtained from GenBank. The whole genome of SARS-CoV TOR2 was divided into overlapping 70-nt segments offset by 25nt. 660 70-mer oligonucleotides were synthesized and printed on glass slides. Sample RNA were transcripted with 6nt random primers, and then amplified and labeled by dUTP-cy3 with a modified random PCR protocol. By using a random PCR amplification strategy in conjunction with the SARS-CoV whole genome viral microarray, we got positive hybridized spots in the whole arrays for the SARS virus(strain BJ01) RNA prepared from cell culture, while negative results were obtained for white blood cell RNA from normal person. 2 PCR products resulting from 2 pairs of specific primers for SARS virus detection recommended by WHO collaborative networks, and 5 cloned gene fragments representing M protein, N protein, S1 protein, S2 protein and a NS protein were used as templates amplified and labeled with the random PCR protocol. The microarray hybridization results showed that sequential positive signal points could be recognized sequentially in their anticipated places. More than 200 clinical specimens have been detected using the microarray hybridization and found the sensitivity is high enough for testing SARS viruses in specimens(data not shown). These results demonstrated that the whole viral genome chip provides a new idea and method for analyzing and testing SAR virus.