摘要
目的 对非典型肺炎的病原体进行分离鉴定 ,为该病的诊断、预防和治疗提供依据。方法 采用细胞培养和乳鼠接种法 ,从非典型肺炎病例标本中分离致病病原体 ,通过电镜形态学、血清学和动物致病性观察及RT PCR扩增与部分基因序列分析 ,对分离的病原体进行鉴定。结果 成功地从死亡病例尸解的肺组织标本和患者鼻咽拭子标本中采用细胞培养法分离出病原体。通过电镜在病变细胞及其培养上清中观察到大量冠状病毒样颗粒。免疫荧光染色检测非典型肺炎患者血清 ,表明分离的病毒与此次流行的非典型肺炎密切相关。分离的病毒可对乳鼠致病 ,并在发病乳鼠的肺组织标本中通过电镜同样观察到冠状病毒样颗粒。从非典型肺炎病例尸解肺组织、传代发病小鼠肺组织及分离物感染的细胞培养物中 ,通过RT PCR可分别扩增出冠状病毒的cDNA片段 ,测序结果显示其核苷酸序列与已知冠状病毒的同源性在 6 0 %左右。结论 从非典型肺炎病例标本中已成功分离出一种新的冠状病毒 ,它与此次流行的非典型肺炎密切相关 。
Objective: To isolate and identify the microbial pathogen of Severe Acute Respiratory Syndrome (SARS), thus to lay the foundation for the diagnosis, prevention and treatment of the SARS. Methods: Cell Cultures and suckling mice were inoculated by the samples from patients with SARS and autoptical tissues. The isolates were identified by morphology, serology, animal experiment, and RT-PCR amplification and partial sequence analysis. The causative association between the isolates and SARS was determined. Results: A possible pathogen of SARS was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with SARS. The isolated microbial agents could cause cytopathic effects on Vero-E6 cells and a large quantity of coronavirus-like particles could be observed in the infected cells and supernatants under transmission electron microcope (TEM). Twenty-three of 26 serum samples from patients with SARS were tested positive by immunofluorescence assay, while 31 of the control serum kept all negative, indicating that the isolated virus was clearly associated with current SARS epidemics. The isolated virus was pathogenic to suckling mice, and coronavirus-like particles could also be seen under TEM. cDNA fragments specific to coronavirus could be amplified by RT-PCR from lung tissues of died patients with SARS, lung tissues of infected mice and cell cultures infected by the isolates, and sequence analysis showed that the homology of the amplified cDNA fragments to the previously known coronaviruses was about 60%～70%. Conclusion: A coronavirus was isolated from the SARS patients. The isolated coronavirus was clearly associated with the current SARS epidemics, and was possibly the key causative agent of SARS. The isolated virus was possibly a noval coronavirus.;
出处
《中国生物工程杂志》
CAS
CSCD
2003年第4期106-112,共7页
China Biotechnology
关键词
非典型肺炎
新型冠状病毒
分离
鉴定
病原体
序列分析
Atypical pneumonia Pathogen Coronavirus Isolation and Identification Sequence analysis
