A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).
A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).
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DOI：
10.3389/fmicb.2014.00395
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发表时间：
2014
影响因子：
5.2
通讯作者：
Schoenfeld TW
中科院分区：
文献类型：
--
作者：
Chander Y;Koelbl J;Puckett J;Moser MJ;Klingele AJ;Liles MR;Carrias A;Mead DA;Schoenfeld TW
满足在低资源环境中提供用于病原体分子检测的护理点（POC）测试的目标对该技术的各个方面提出了严格的要求。OmniAmp DNA聚合酶（Pol）是一种耐热病毒酶，通过克服等温扩增的重要障碍，使真正的POC能够在临床或现场使用。在本文中，我们描述了多个优势的Omnitrile Pol作为等温扩增酶，并提供了其使用的环介导等温扩增（LAMP）的病原体检测的例子。Omnitriol的固有逆转录酶活性允许在RT-LAMP中对RNA靶标进行单酶检测。核酸扩增的常见方法对样品污染物高度敏感，需要与POC或现场使用不相容的精细核酸纯化方案。发现OmniblePol受某些粗样品制备液中典型的全血组分的抑制较小。此外，与替代DNA聚合酶（Bst）和逆转录酶相比，酶的热稳定性允许在扩增之前立即预处理完全反应混合物，这有助于扩增高度结构化的基因组区域。与Bst相比，Omnitrile Pol具有更快的结果生成时间，特别是对于更稀的模板。由于缺乏制冷，现场环境中的分子诊断可能具有挑战性。Omnitrile Pol的稳定性与干燥形式兼容，可在环境温度下长期储存。现场可操作性的最终要求是与通常可用的仪器兼容，或者在其他情况下，与需要最少培训或功率的简单、廉价、便携式检测模式兼容。扩增产物的检测显示使用横向流动条和实时PCR仪器上的分析。这项研究的结果表明，Omnitrile Pol非常适合低资源的病原体分子检测。
Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.
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影响因子：
3.7
作者：
Myers FB;Henrikson RH;Bone JM;Lee LP
通讯作者：
Lee LP
影响因子：
3
作者：
Fu, Shijun;Qu, Guanggang;Shen, Zhiqiang
通讯作者：
Shen, Zhiqiang
影响因子：
3.3
作者：
Nagamine, K;Hase, T;Notomi, T
通讯作者：
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影响因子：
6.1
作者：
Stedtfeld, Robert D.;Tourlousse, Dieter M.;Hashsham, Syed A.
通讯作者：
Hashsham, Syed A.
DOI：
10.1016/j.diagmicrobio.2010.08.026
发表时间：
2011-02-01
影响因子：
2.9
作者：
Njiru, Zablon Kithinji
通讯作者：
Njiru, Zablon Kithinji