基于多重置换扩增技术的RNA病毒基因组扩增方法研究  

Investigation of RNA Viral Genome Amplification by Multiple Displacement Amplification Technique

作　　者：庞正[1] 李建东[1] 李川[1] 梁米芳[1] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒性出血热室,北京102206

出　　处：《病毒学报》2013年第4期432-436,共5页Chinese Journal of Virology

基　　金：传染病防治重大专项-重大传染病应急处置检测平台(No.2011ZX10004-001,2013ZX10004-001)

摘　　要：为了便于新发或罕见病毒性传染病的筛查检测,本研究利用多重置换扩增技术,以负链RNA病毒—发热伴血小板减少综合征病毒和正链RNA病毒—登革病毒为模拟样本探索临床样本中RNA病毒基因组非特异性扩增方法。研究中通过梯度稀释的RNA病毒模拟样本中可能存在的不同丰度的病原体,样本核酸依次加工成单链cDNA、双链cDNA、T4DNA连接酶处理后的双链cDNA以及添加外源辅助RNA后合成并连接的双链cDNA形式,然后进行Phi29DNA聚合酶等温扩增,使用荧光定量PCR方法比较各种方法对RNA病毒核酸扩增的影响。结果显示,对于不同类型的RNA病毒模拟标本,多重置换扩增对于单链及双链cDNA的扩增效果有限,而双链cDNA经DNA连接酶处理后的扩增能达到6×103倍;在cDNA合成过程中加入外源辅助RNA,模拟样本中病毒基因组的扩增可达2×105倍,尤其是对含有低丰度病原体的模拟样本扩增效果的改善更为明显。本研究摸索建立了基于多重置换扩增技术的RNA病毒基因组扩增方法,能够对样本中低丰度RNA病毒基因组实现有效扩增,可满足开展多种病原体筛查检测的需求。;In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negativestrand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple dis- placement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the am- plification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of doublestrand cDNA templates treated with ligation could be up to 6×10^5, even 2 ×10^5 when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthe- size adequate viral genome for multiplex pathogens detection.

关 键 词：多重置换扩增 Phi29DNA聚合酶 RNA病毒 

分 类 号：R373.9[医药卫生—病原生物学]