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 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.

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

“…While tHDA is a simple, rapid amplification method, many of the described detection platforms still require extensive preparation and extraction steps for unprocessed cell samples prior to amplification. The Kamei group described a one-pot isothermal platform that incorporates cell lysis, DNA extraction, and tHDA utilizing an aqueous two-phase system (ATPS) ( Cheung et al, 2018 ). A Triton X-100 micellar ATPS was combined with tHDA reagents and a cell suspension containing intact E. coli cells.…”

“…Whole cell samples were used in these experiments. Reprinted by permission from Springer Nature: Springer Nature, Analytical and Bioanalytical Chemistry ( Cheung et al, 2018 ), Copyright 2018. …”

Advanced Signal-Amplification Strategies for Paper-Based Analytical Devices: A Comprehensive Review

“…Conventionally, polymerase chain reaction (PCR) is the technique extensively used to rapidly process millions to billions of DNA samples, but it requires precise temperature control for reactions, and hence, to date has been unsuccessfully applied to PADs [ 149 , 150 ]. Alternatively, various signal amplification isothermal methods have been tried for PAD development, including rolling circle amplification (RCA) [ 147 , 151 , 152 , 153 , 154 , 155 ], loop-mediated isothermal amplification (LAMP) [ 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 ], strand-displacement amplification (SDA) [ 167 ], recombinase polymerase amplification (RPA) [ 168 , 169 , 170 , 171 ], helicase dependent amplification (HDA) [ 172 , 173 , 174 ], and nucleic acid sequence-based amplification (NASBA) [ 175 ].…”

Nucleic acid extraction: Fundamentals of sample preparation methodologies, current advancements, and future endeavors

“…[35] The procedure. [37] After lysing the cells in the detergent phase, DNA migrated to the tHDA buffer phase where it was amplified. This method has the potential for POC diagnostics if visual detection is utilized as only a heat block would be required for analysis.…”