随机引物聚合酶链反应法鉴定2例无菌性脑炎的未知病毒  

Detection of Unknown Virus from 2 Case of Aseptic Meningitis Epidemic Using Random Primer PCR Assay

作　　者：陈寅[1] 严菊英[1] 卢亦愚[1] 茅海燕[1] 张严峻[1] 

机构地区：[1]浙江省疾病预防控制中心,杭州310051

出　　处：《中国疫苗和免疫》2009年第4期341-344,共4页Chinese Journal of Vaccines and Immunization

摘　　要：目的建立一种可应用于准确检测未知病毒的分子生物学方法。方法2例无菌性脑炎患者的脑脊液,接种人横纹肌肉瘤细胞,收集感染细胞液,将细胞液离心去除细胞碎片,超速离心沉淀病毒颗粒后,用脱氧核糖核酸酶处理,提取核酸后,用随机引物进行聚合酶链反应(Polymerase Chain Reaction,PCR)扩增,将扩增产物克隆至pGEM-TEsay载体中测序,序列通过BLAST(Basic Local Alignment Search Tool)最终确定未知病毒。结果经BLAST比对证明,该病毒是人类肠道致细胞病变孤儿病毒4型。结论成功建立了一种基于随机引物PCR的未知病毒鉴定方法,为快速检测不明原因疾病和新发传染病病原提供了新的技术方法。Objective To develop a molecular biology method which can be applied to detect unknown virus fast and accurately. Methods The samples were separated from human cerebrospinal fluid by infected human rhabdomyosarcoma cells. After centrifugation and discarding the cell debris,the samples were subjected to ultracentrifugation to precipitate viral particles. The DNase treated samples were used to extract viral nucleic acids. The product of random RT-PCR amplification was cloned into pGEM-T Easy vector and sequenced. Sequence similarities were identified with the BLAST programs. Results The BLAST results showed that the viral agent was ECHO4 strain. Conclusion A random primer PCR method was established,which can be used to identify novel viruses. This research provides a random amplification method for effective detection of unknown viruses or emerging infectious pathogens.

关 键 词：无菌性脑炎 未知病毒 随机引物扩增 人类肠道致细胞病变孤儿病毒 

分 类 号：R373.2[医药卫生—病原生物学]