Abstract
The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings.
Highlights
Polymerase chain reaction (PCR) is widely considered to be the gold standard for sensitive and specific diagnosis of many infectious diseases
We explored the feasibility of using body heat to incubate recombinase polymerase amplification (RPA) reactions for amplification of HIV-1 DNA
The temperature of mock RPA reactions was measured at various body locations to estimate the temperature that an RPA reaction would reach if incubated using body heat
Summary
Polymerase chain reaction (PCR) is widely considered to be the gold standard for sensitive and specific diagnosis of many infectious diseases. PCR requires access to expensive thermal cycling equipment that is frequently unavailable in low-resource settings where the infectious disease burden is greatest. A number of platforms have been developed to amplify nucleic acids at a single temperature, alleviating the need for thermal cycling equipment [5,6,7,8,9]. Because isothermal amplification methods require only a single temperature, these platforms can be implemented using a simple, fixed-temperature heater, which costs at least an order of magnitude less than a thermal cycler [10]. The design constraints for such heaters, such as temperature set-point and stability, highly depend on the intended isothermal amplification platform and ambient temperature range
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