A one-pot, isothermal DNA sample preparation and amplification platform utilizing aqueous two-phase systems

Abstract

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 102 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.

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Acknowledgments

This work was supported by a grant from the UCLA School of Dentistry.

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Correspondence to Daniel T. Kamei.

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Conflict of interest

B.M. Wu and D.T. Kamei are co-founders of the company Phase Diagnostics that intends on commercializing this core technology. In addition to being co-founders and having equity in Phase Diagnostics, they are members of the Board of Directors and the Scientific Advisory Board. Phase Diagnostics has licensed this intellectual property from the UC Regents. D.T. Kamei, B.M. Wu, and S.F. Cheung are co-inventors on the patent application regarding this work. D.T. Kamei also has had and still has small business grants with Phase Diagnostics funded by the National Institutes of Health and the National Science Foundation. M.F. Yee and N.K. Le have no conflict of interest.

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Cheung, S.F., Yee, M.F., Le, N.K. et al. A one-pot, isothermal DNA sample preparation and amplification platform utilizing aqueous two-phase systems. Anal Bioanal Chem 410, 5255–5263 (2018). https://doi.org/10.1007/s00216-018-1178-4

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Keywords

  • Nucleic acid amplification test (NAAT)
  • Isothermal
  • Thermophilic helicase-dependent amplification (tHDA)
  • Aqueous two-phase systems (ATPSs)
  • Triton X-100