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为查找引起广州地区流行的严重急性呼吸综合征(SARS)的病原体,采集患者漱口液及尸解标本,用组织培养法接种人胚肺细胞、MDCK细胞、Hep-2细胞和鸡胚分离病毒,用间接免疫荧光法检测患者恢复期血清IgG抗体,确定分离的病原是SARS的主要病因,再用套式RT-PCR、免疫电镜法鉴定病原。结果用人胚肺、Hep-2细胞在75份漱口液和3例尸解组织中分离出13株病原体,经套式RT-PCR扩增出110bp的特异产物,经测序证实为冠状病毒。制备冠状病毒的抗原,检测30份SARS病人恢复期血,其中26份血清IgG抗体阳性。同时检测30份普通发热病人血清作对照,IgG抗体全部阴性。由此证明,经组织培养分离到的病原体是引起SARS的致病因子,用分子生物学方法测序后证实为冠状病毒。
To find out the pathogen of severe acute respiratory syndrome (SARS) causing epidemic in Guangzhou, the patients’ mouthwash and autopsy specimens were collected, and the human embryo lung cells, MDCK cells, Hep-2 cells and chicken embryos were isolated by tissue culture , Indirect immunofluorescence assay of serum IgG antibody in convalescent patients to determine the pathogen is the main cause of SARS, and then use nested RT-PCR, immunoelectron microscopy identification of pathogens. Results Thirteen pathogens were isolated from human embryo lung and Hep-2 cells in 75 mouthwashes and 3 autopsy tissues. The specific products of 110 bp were amplified by nested RT-PCR and confirmed to be coronavirus by sequencing. The coronavirus antigens were prepared and 30 convalescent blood samples were collected from SARS patients, of which 26 were positive for IgG antibodies. At the same time detection of 30 common fever patients serum as a control, all negative IgG antibodies. Thus, it was proved that the pathogen isolated from tissue culture was the causative agent of SARS and confirmed as a coronavirus by molecular biology method.