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 screening of new or rare viral infectious diseases, this study explored the clinical use of multiple replacement amplification (PCR) with negative-strand RNA virus-fever with thrombocytopenia syndrome virus and positive-strand RNA virus-dengue virus Non-specific amplification of RNA viral genomes in samples. In this study, samples of different abundance of pathogens may be simulated by gradient dilution RNA viruses. The sample nucleic acids are sequentially processed into single-stranded cDNA, double-stranded cDNA, double-stranded cDNA treated with T4 DNA ligase, and the addition of exogenous helper RNA And ligated double-stranded cDNA. Then, Phi29 DNA polymerase was amplified by isothermal amplification. The effects of various methods on nucleic acid amplification of RNA virus were compared by fluorescence quantitative PCR. The results showed that for different types of RNA viruses simulated mimotope amplification amplification for single-stranded and double-stranded cDNA amplification effect is limited, and double-stranded cDNA by DNA ligase amplification can reach 6 × 103 times; in In the process of cDNA synthesis, the exogenous helper RNA is added and the amplification of the virus genome in the simulated sample is up to 2 × 10 5 times, especially for the simulation samples containing low abundance pathogens. This study explored the RNA amplification method based on multiple replacement amplification to amplify the genome of low-abundance RNA viruses in samples and meet the needs of multiple pathogen screening tests.