SARS冠状病毒的分离培养与鉴定  

Isolation and Identification of SARS-Coronavirus

作　　者：江丽芳[1] 赵卫[2] 晏辉钧[1] 方丹云[1] 周经姣[1] 龙北国[2] 吴义芳[1] 周俊梅[1] 梁瑜[1] 张文炳[2] 吴强[1] 郭辉玉[1] 

机构地区：[1]中山大学中山医学院微生物学教研室,广东广州510080 [2]第一军医大学热卫系微生物学教研室,广东广州510515

出　　处：《中国病毒学》2003年第6期544-547,共4页Virologica Sinica

基　　金：国家自然科学基金(30340013);广东省科技攻关项目

摘　　要：采集急性期病人的咽拭子或漱口液,用Vero、Vero E6、MDCK、Hela、Hep-2等传代细胞,人胚肺二倍体细胞(HEL)和人胚肺(HP)细胞分离培养严重急性呼吸系统综合症(SARS)的病原体。结果用Vero、Vero E6、MDCK和HP细胞从标本中分离到一株病毒。间接免疫荧光试验发现,恢复期病人血清可与所分离的病毒起反应,在胞膜和胞浆中出现翠绿色荧光;中和试验结果表明,恢复期病人血清能中和病毒对细胞的致细胞病变作用:电镜下可观察到冠状病毒样颗粒:RT-PCR法可扩增到冠状病毒特异性基因片段,且其核苷酸序列与国内外发表的SARS冠状病毒(SARS-Cov)相应的基因序列相符,同源性达到100％。从传染性非典型肺炎病人的漱口液中分离到SARS冠状病毒,这种病毒与传染性非典型肺炎密切相关。To isolate and identify the etiologic agent of severe acute respiratory syndrome(SARS), the throat swabs and gargle of patients with SARS were collected. We inoculated the specimens onto a number of cell lines including Vero, Vero E6, MDCK, Hela, Hep-2, HP and HEL cells. The isolates were identified by thin-section electron-microscopy, indirect-immunofluorescence, neutralization test, RT-PCR and nucleotide sequence analysis. Using cell culture techniques, a coronavirus was isolated from patient with SARS. Four cell lines, Vero, Vero E6, HP and MDCK cells, showed cytopathic effect. The infected cells showed strong cytoplasmic and membranous staining with a convalescent-phase serum from the patients with SARS in an indirect-immunofluorescence staining. The cytopathic effect of the viruses clould been neutralized by convalescent serum. Under electron-microscopy, a large number of coronavirus-like particles were obsewed in the infected cells. With specific primers, the specific cDNA fragments were amplified by reverse transcription-polymerase chain reaction(RT-PCR) from infected cells and the nucleotide sequence analysis showed that homology of the amplified fragments to the SARS-coronavirus (SARS-CoV) previously reported was 100%. A SARS-CoV was isolated from patients with SARS. This virus was closely associated with SARS.

关 键 词：传染性非典型肺炎 SARS冠状病毒 分离 培养 鉴定 序列分析 

分 类 号：R373[医药卫生—病原生物学] Q939.4[医药卫生—基础医学]