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基于 phi29 的全基因组扩增与全转录组扩增在登革病毒中的比较。

Comparison of phi29-based whole genome amplification and whole transcriptome amplification in dengue virus.

机构信息

Graduate Program in Immunology, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Division of Bioinformatics and Data Management for Research, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Center for Emerging and Neglected Infectious Diseases, Mahidol University, Bangkok, Thailand.

出版信息

J Virol Methods. 2014 Jan;195:141-7. doi: 10.1016/j.jviromet.2013.10.005. Epub 2013 Oct 12.

Abstract

Dengue virus is responsible for 50-100 million new infections annually worldwide. The virus uses error-prone RNA polymerase during genome replication in a host, resulting in the formation of closely related viruses known as quasispecies. The availability of next-generation sequencing technology provides opportunities to analyze viral quasispecies. Before analysis, it is crucial to increase the amount of DNA because of the limited amounts of viral genomic material that can be isolated from a patient. However, using specific primers may overlook the occurrence of possible variations at primer binding sites. To address this problem, the performance of two sequence-independent amplification methods was compared for whole genome amplification (WGA): phi29 DNA polymerase-based WGA and whole transcriptome amplification (WTA). Both methods have the ability to provide complete coverage of the dengue genome from template amounts as low as 1 ng. However, WTA showed greater efficiency in terms of yield (WTA: ~10 μg; phi29-based WGA: ~500 ng) and lower amplification bias. In conclusion, the WTA amplification kit was shown to perform substantially better than phi29 DNA polymerase-based WGA in terms of both final concentration and amplification bias in amplifying small genomes, such as that of the dengue virus.

摘要

登革热病毒每年在全球范围内导致 5000 万至 1 亿例新感染。该病毒在宿主中利用易错 RNA 聚合酶进行基因组复制,导致形成称为准种的密切相关的病毒。下一代测序技术的出现为分析病毒准种提供了机会。在分析之前,由于从患者中分离出的病毒基因组物质数量有限,增加 DNA 的数量至关重要。然而,使用特定引物可能会忽略引物结合位点可能发生的变异。为了解决这个问题,比较了两种序列非依赖性扩增方法(phi29 DNA 聚合酶基 WGA 和全转录组扩增(WTA))用于全基因组扩增(WGA):phi29 DNA 聚合酶基 WGA 和全转录组扩增(WTA)。这两种方法都能够从低至 1ng 的模板量提供对登革热基因组的完整覆盖。然而,WTA 在产量方面表现出更高的效率(WTA:10μg;phi29 基 WGA:500ng)和更低的扩增偏差。总之,与 phi29 DNA 聚合酶基 WGA 相比,WTA 扩增试剂盒在扩增小基因组(如登革热病毒)时,无论是最终浓度还是扩增偏差,都表现出明显更好的性能。

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