【摘要】 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。更多还原

【Abstract】 Objective:To establish pathogenic detection methods of Severe Acute Resqiratory Syndrome(SARS) and to provide a basis for diagnose and field prevention and control for SARS.Methods: Detection of the nucleic acid of SARS-CoV by RT-Nested-PCR and real-time PCR;detection of the IgM antibody to SARS-CoV by ELISA;the specimens which have shown PCR or ELISA positive result were observed for SARS-CoV by electronic microscope;detection of the nucleic acid of Coronavirus by PCR using genus-specific degenerate primers.For differential diagnose,IgM antibodies to nine frequent pathogens of respiratory such as mycoplasma pneumoniae and etc.were tested by IFA,the nucleic acid of Influenza A or B,and Legionella pneumophila were examined by PCR amplification. Results: There were total of 506 clinical samples were detected.In 2003,7 SARS cases were reported with clinical diagnose,5 of which were positive with laboratory detection;in 132 samples of different types,8 positives were detected by using RT-Nested-PCR.Four PCR products(108bp) of the positives were sequenced and compared with SARS-CoV in the GenBank by using BLAST server,it showed 100% homology.Three of 30 clinical samples in different types and 4 of 101 serum samples were found to be positive by real-time PCR and ELISA,respectively.SARS-Cov-like particles were observed in different type clinical samples in three SARS cases.There are 75 serum samples were detected for IgM antibody against 9 respiratory pathogens respectively by IFA.The results showed that 9 were positive to Legionella pneumonia,11 were to Influenza B,2 were to both Legionella pneumonia and Influenza B,and the rest were negative.Twenty-one samples from patients who have unknown cause of fever disease were detected by Coronavirus-specific PCR using degenerate primers and 2 throat swabs were found positive.Fifteen throat-washing samples and 15 urine samples were detected for Legionella pneumonia, and 25 throat-washing samples were detected for Influenza A and B,the results were all negative.Conclusion: The detective result using the methods above mentioned has quite accordant with that of clinical diagnosis and PCR method has high specificity for SARS-CoV.更多还原