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目的 探讨冠状病毒感染与传染性非典型肺炎发病的关系 ,分析冠状病毒基因序列变异的临床意义 ,建立诊断冠状病毒变异株感染的分子生物学方法。方法 收集非典型肺炎 (SARS)患者的不同临床标本 ,采用PCR技术分别进行衣原体、甲肺炎病毒以及冠状病毒等的基因检测。以冠状病毒相对保守的依赖RNA的RNA多聚酶基因为靶基因 ,采用NestedRT PCR技术从非典型肺炎患者的鼻咽吸取物标本和漱喉液标本中扩增出冠状病毒基因 ,将PCR产物直接克隆到T载体 ,进行序列测定 ,进一步比较不同分离株间的核苷酸和氨基酸序列同源性。结果 在 2 7例非典型肺炎患者中检出冠状病毒基因阳性 6例 ,阳性率为 2 2 2 %。而在 15例健康对照中 ,全部阴性。采用BLAST软件将所获得的冠状病毒的基因序列与基因库 (GenBank)登记的序列比较 ,结果显示本次分离克隆的冠状病毒基因与既往报道的所有冠状病毒在核苷酸水平没有相关性 ,核苷酸同源性最高不超过 4 0 %。但在氨基酸水平的同源性为 80 %左右 (70 %～ 82 % )。从不同患者中分离的冠状病毒基因之间在核苷酸水平的同源性在 98%以上。与刚刚公布的加拿大、中国香港、英国等SARS冠状病毒的核苷酸序列同源性在 99%以上。结论 相当部分非典型肺炎患者存在冠状病毒的感染 ,提示本次的非典型肺
Objective To investigate the relationship between coronavirus (SAR) infection and the development of SARS, and to analyze the clinical significance of variation in the sequence of the coronavirus gene and to establish a molecular biological method for the diagnosis of coronavirus variant infection. Methods Different clinical specimens of patients with atypical pneumonia (SARS) were collected. PCR was used to detect the genes of Chlamydia, A pneumonia and Coronavirus. The RNAi-based RNA polymerase gene, which is a relatively conserved coronavirus, was used as a target gene. The coronavirus gene was amplified from specimens of nasopharyngeal aspirates and lachrymal samples from patients with atypical pneumonia by NestedRT PCR. The PCR products were directly cloned into T vector and sequenced to further compare the nucleotide and amino acid sequence homologies among different isolates. Results Of the 27 patients with SARS, 6 were positive for coronavirus. The positive rate was 22.2%. In 15 healthy controls, all were negative. The BLAST software was used to compare the sequence of the obtained coronavirus with that registered in GenBank. The results showed that there was no correlation between the coronavirus gene of this isolate and all the previously reported coronaviruses at nucleotide level. The highest nucleotide homology does not exceed 40%. However, the homology at the amino acid level is about 80% (70% -82%). The homology between the coronavirus genes isolated from different patients at the nucleotide level is above 98%. With just published in Canada, Hong Kong, China, Britain and other SARS-CoV nucleotide sequence homology of more than 99%. Conclusion A significant proportion of patients with SARS have coronavirus infection, suggesting that this atypical pneumonia