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首页> 外文期刊>BMC Biotechnology >Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection
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Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

机译：多种策略可提高等温核酸扩增的灵敏度，速度和鲁棒性，以快速检测病原体

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Background 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). Results 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). Conclusions 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.
机译：背景技术在过去的几十年中，分子诊断技术（基于传统的PCR或等温扩增技术）的迅速发展满足了对快速准确的测试的需求。尽管等温扩增技术的优点是对仪器的成本要求低，但是灵敏度，速度和耐用性的进一步提高是快速检测病原体，尤其是在现场诊断中应用的先决条件。在这里，我们描述和探索了几种改善等温技术之一的方法，即解旋酶依赖性扩增（HDA）。结果采取了多种策略来改善等温扩增的整体性能：限制性核酸内切酶介导的DNA解旋酶归巢，大分子拥挤剂和反应酶混合物的优化。将组合所有策略的效果与单个策略的效果进行了比较。通过上述所有方法，我们可以使用几乎没有仪器的检测平台（BESt？盒）在短短20分钟的扩增中检测出50株淋病奈瑟氏球菌DNA。结论本概念验证研究中涉及的策略与昂贵的设备无关，并且不限于特定的引物，靶标或检测形式。但是，它们在测定性能上有很大的不同。其中一些可以调整并应用于其他形式的核酸扩增。此外，通过最大程度地模拟自然条件来改善体外测定的策略可能在开发分子测定的一般领域中有用。还开发了一种用于淋病奈瑟氏球菌的新的快速分子测定法，它在现场诊断中具有巨大的潜力。

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