采用随机聚合酶链反应方法对临床样本中未知核糖核酸病毒检测的研究  

Study on the Detection of the Unknown Ribonucleic Acid Viruses in the Clinical Specimens Using Random Polymerase Chain Reaction Assay

作　　者：黄崴[1] 卢亦愚[2] 冯燕[1] 陈寅[2] 徐昌平[2] 

机构地区：[1]温州医学院检验医学院暨生命科学院,浙江温州325035 [2]浙江省疾病预防控制中心,杭州310051

出　　处：《中国疫苗和免疫》2011年第3期237-241,264,共6页Chinese Journal of Vaccines and Immunization

基　　金：浙江省公共卫生检验检测重点学科群(XKQ-009-003)

摘　　要：目的探索一种不依赖于病毒培养,直接对临床样本中未知核糖核酸(Ribonucleic Acid,RNA)病毒作出鉴定的分子生物学方法。方法对经前处理后的临床样本提取病毒RNA,进行随机聚合酶链反应(Polymerase Chain Reaction,PCR)扩增,通过对扩增产物的纯化、克隆、测序以及序列的在线比对,根据同源性确定核酸片段的来源,并最终实现对未知病毒的鉴定。对临床样本的前处理以及前处理方法的优化,即通过冻融、离心、过滤、聚乙二醇浓缩,以及核糖核酸酶和脱氧核糖核酸酶的处理,尽可能地去除样本中的无关核酸,以增大病毒核酸在样本中所占比例,提高阳性检出率。结果通过对样本前处理方法的改进,从病毒载量为4.8×103Copy/ml(拷贝/毫升)的含漱液样本中,以70%的阳性克隆率检测出未知RNA病毒。结论所建立的方法能提高对RNA病毒鉴定的阳性率,这将在不明原因传染病病原体的鉴别诊断中,发挥重要的作用。Objective To establish a tissue culture-independent assay to directly identify the unknown ribonucleic acid（RNA）viruses in clinical specimens. Methods The nucleic acid extracted from the clinical specimens after pre-treatment was amplified by random-polymerase chain reaction（PCR）. The PCR products were purified, cloned, sequenced. The obtained sequences were then submitted to basic local alignment search tool（BLAST）. According to the homology,sources of the nucleic acid fragments could be determined and the unknown viruses could be successfully identified. The pre-processing for the clinical specimens,including freezing and thawing,centrifugation,filtration,concentration with polyethylene glycol,treatment with ribonuclease and deoxyribonuclease,were adopted and optimized to remove the unrelated nuclelic acid as many as possible,which can increase the ratio of the target nucleic acid and improve the detection rate of target viruses. Results Through the improvement on the preprocessing of clinical specimens,the unknown RNA viruses were detected in 70% rate of viral positive clones,of which the viral load was 4.8×103 copies/ml. Conclusion The culture-independent random PCRbased assay developed in this study greatly increases the detection rate of the unknown RNA viruses,which could play an important role in the identification on the pathogens of the unknown infectious disease.

关 键 词：未知核糖核酸病毒 鉴定 样本前处理 随机聚合酶链反应扩增 

分 类 号：R373[医药卫生—病原生物学]