Department of Bioengineering, University of California, 5121 Engineering V, 420 Westwood Plaza, Los Angeles, CA, 90095, USA.
Division of Advanced Prosthodontics & Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry, Los Angeles, CA, 90095, USA.
Anal Bioanal Chem. 2018 Aug;410(21):5255-5263. doi: 10.1007/s00216-018-1178-4. Epub 2018 Jun 8.
Infectious diseases remain one of the major causes of death worldwide in developing countries. While screening via conventional polymerase chain reaction (PCR) is the gold standard in laboratory testing, its limited applications at the point-of-care have prompted the development of more portable nucleic acid detection systems. These include isothermal DNA amplification techniques, which are less equipment-intensive than PCR. Unfortunately, these techniques still require extensive sample preparation, limiting user accessibility. In this study, we introduce a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar aqueous two-phase system (ATPS) to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to amplify and detect a target gene from whole-cell samples containing as low as 10 cfu/mL, and is the first known application of ATPSs to isothermal DNA amplification. This system's ease-of-use and sensitivity underlie its potential as a point-of-care diagnostic platform to detect for infectious diseases. Graphical abstract ᅟ.
传染病仍然是发展中国家全球死亡的主要原因之一。虽然传统聚合酶链反应 (PCR) 检测是实验室检测的金标准，但由于其在即时检测点的应用有限，促使人们开发了更便携的核酸检测系统。其中包括等温 DNA 扩增技术，它比 PCR 的设备要求更低。不幸的是，这些技术仍然需要广泛的样品制备，限制了用户的可及性。在本研究中，我们介绍了一种新系统，该系统将嗜热解旋酶依赖性扩增 (tHDA) 与 Triton X-100 胶束双水相系统 (ATPS) 相结合，在一个简单的一步法中实现细胞裂解、裂解产物处理和增强的核酸扩增。组合的一锅法系统能够从含有低至 10 cfu/mL 的全细胞样品中扩增和检测靶基因，并且是首次已知将 ATPS 应用于等温 DNA 扩增。该系统的易用性和灵敏度为即时检测点诊断平台检测传染病提供了潜力。
