Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.
Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.
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DOI：
10.1186/1472-6750-11-50
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发表时间：
2011-05-11
影响因子：
3.5
通讯作者：
Kong H
中科院分区：
文献类型：
--
作者：
Tong Y;Lemieux B;Kong H
在过去的几十年中，分子诊断（基于传统的PCR或等温扩增技术）的快速增长满足了快速和准确检测的需求。尽管等温扩增技术具有对仪器的低成本要求的优点，但进一步提高灵敏度、速度和鲁棒性是应用于快速病原体检测，特别是在护理点诊断中的先决条件。在这里，我们描述和探讨几种策略，以改善等温技术之一，解旋酶依赖性扩增（HDA）。采用限制性内切酶介导的DNA解旋酶归巢、大分子拥挤剂和优化反应酶混合物等多种策略来提高等温扩增的整体性能。比较了组合策略与单独策略的效果。通过上述所有方法，我们能够使用几乎无仪器的检测平台（BESt™盒）在仅20分钟的扩增中检测50个拷贝的淋病奈瑟菌DNA。本概念验证研究中提出的策略不依赖于昂贵的设备，并且不限于特定的引物、靶标或检测形式。然而，它们在测定性能方面存在很大差异。它们中的一些可以被调整并应用于核酸扩增的其他形式。此外，通过最大限度地模拟自然条件来改进体外分析的策略可能在开发分子分析的一般领域中是有用的。还开发了一种新的淋病奈瑟菌快速分子检测方法，该方法具有很大的潜力，可用于即时诊断。
In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA). Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BESt™ cassette). The strategies addressed in this proof-of-concept study are independent of expensive equipments, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.
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通讯作者：
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通讯作者：
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通讯作者：
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