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目的:建立传染性非典型肺炎(SARS)实验室检测方法,为SARS病例诊断和现场防治提供依据。方法:用逆转录套式PCR、实时荧光定量PCR检测SARS病毒核酸;酶联免疫法检测血清标本SARS病毒IgM抗体;部分PCR检测阳性或SARS病毒IgM抗体阳性病例的标本进行直接电镜观察;简并引物PCR检测冠状病毒属病毒核酸;鉴别诊断IFA检测血清中肺炎支原体等9种常见呼吸道感染病原IgM抗体,PCR检测军团菌以及甲、乙型流感病毒核酸。结果:检测各类临床标本506份。2003年全省报告临床诊断病例7例,5例检测结果阳性;逆转录套式PCR检测各类标本132份,阳性8份;对其中4份PCR扩增产物(108bp)进行了序列分析,与SARS病毒序列100%同源;实时荧光定量PCR检测各类标本30份,阳性3份;酶联免疫法检测血清标本101份,SARS病毒IgM抗体阳性4份;3个诊断病例的各类标本电镜检查发现冠状病毒样颗粒;IFA检测血清标本75份,嗜肺军团菌IgM抗体阳性9份,乙型流感IgM抗体阳性11份,两者同时阳性3份,其他病原体IgM抗体均为阴性。不明发热病例的标本21份简并引物PCR检测冠状病毒,2份咽拭子标本阳性;漱口液、尿液标本各15份PCR检测军团菌、漱口液25份PCR检测甲、乙型流感病毒结果均为阴性。结论:本研究建立的检测方法与临床诊断有比较好的符合性,对于临床诊断和防治工作具有重要的参考价值。PCR检测SARS-CoV特异性较好,而敏感性有待提高。
Objective: To establish a laboratory test for SARS and provide a basis for the diagnosis and prevention of SARS cases. Methods: SARS virus nucleic acid was detected by reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative polymerase chain reaction (PCR); Serum samples of SARS virus IgM were detected by enzyme-linked immunosorbent assay (ELISA); some specimens were positive by PCR or SARS virus IgM antibody positive; Primer PCR detection of coronavirus nucleic acid; differential diagnosis IFA detection of serum Mycoplasma pneumoniae and other 9 kinds of common respiratory tract infection IgM antibodies, PCR detection of Legionella and influenza A and B virus nucleic acid. Results: All kinds of clinical specimens were tested 506. In 2003, 7 cases of clinical diagnosis were reported in the province, and 5 cases were positive. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect 132 samples of all kinds and 8 were positive. Four PCR products (108bp) were sequenced, SARS virus sequence was 100% homologous; 30 samples were positive and 3 were positive by real-time fluorescence quantitative PCR; 101 serum samples were detected by enzyme-linked immunosorbent assay and 4 were positive by SARS virus IgM antibody; Coronavirus-like particles were detected by IFA assay. 75 serum samples from IFA test, 9 positive IgM antibodies against Legionella pneumophila and 11 positive IgM antibodies against influenza B were obtained. Both of them were positive at the same time, and all other IgM antibodies were negative. 21 samples of unidentified febrile cases were detected by PCR with degenerate primers and 2 samples of throat swabs, and 15 samples of mouthwash and urine samples were detected by PCR. Virus results were negative. Conclusion: The detection method established in this study has good conformance with clinical diagnosis, which has important reference value for clinical diagnosis and prevention and treatment. PCR detection of SARS-CoV specificity is good, and the sensitivity needs to be improved.