Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV
Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV
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DOI：
10.1016/j.jviromet.2015.02.004
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发表时间：
2015-04-01
影响因子：
3.1
通讯作者：
Bialasiewicz, Seweryn
中科院分区：
文献类型：
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作者：
Rockett, Rebecca;Barraclough, Katherine A.;Bialasiewicz, Seweryn
最近发现了 11 种新的人类多瘤病毒，但对于其中大多数病毒，人们对其生物学和临床影响知之甚少。滚环扩增 (RCA) 因其对环状 DNA 的高保真扩增而成为环状多瘤病毒基因组扩增的理想方法。在这项研究中，开发了一种改良的 RCA 方法来选择性扩增一系列多瘤病毒基因组。初步评估显示，多重温度分级反应曲线在人类 DNA 背景下扩增 BK 多瘤病毒时具有最佳产量和灵敏度，每次反应的病毒基因组拷贝数仅为 10 个，病毒基因组数量增加高达 1 x 10(8) 倍。此外，该方法被证明比基于随机六聚体的标准 RCA 更灵敏，产量高 200 倍。该方法应用于其他新型人类多瘤病毒，结果显示从低病毒载量阳性临床样本中成功扩增了 TSPyV、HPyV6、HPyV7 和 STLPyV，病毒基因组富集范围为 1 x 10(8) 至 1 x 10(10)。这种定向 RCA 方法可用于从竞争性非特异性 DNA 背景中选择性扩增其他低拷贝多瘤病毒基因组，并且是进一步研究快速扩张的多瘤病毒科的有用工具。 (C) 2015 Elsevier B.V. 保留所有权利。
Eleven new human polyomaviruses have been recently discovered, yet for most of these viruses, little is known of their biology and clinical impact. Rolling circle amplification (RCA) is an ideal method for the amplification of the circular polyomavirus genome due to its high fidelity amplification of circular DNA. In this study, a modified RCA method was developed to selectively amplify a range of polyomavirus genomes. Initial evaluation showed a multiplexed temperature-graded reaction profile gave the best yield and sensitivity in amplifying BK polyomavirus in a background of human DNA, with up to 1 x 10(8)-fold increases in viral genomes from as little as 10 genome copies per reaction. Furthermore, the method proved to be more sensitive and provided a 200-fold greater yield than that of random hexamers based standard RCA. Application of the method to other novel human polyomaviruses showed successful amplification of TSPyV, HPyV6, HPyV7, and STLPyV from low-viral load positive clinical samples, with viral genome enrichment ranging from 1 x 10(8) up to 1 x 10(10). This directed RCA method can be applied to selectively amplify other low-copy polyomaviral genomes from a background of competing non-specific DNA, and is a useful tool in further research into the rapidly expanding Polyomaviridae family. (C) 2015 Elsevier B.V. All rights reserved.