Body Heat

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.

Body Heat

One such isothermal platform, recombinase polymerase amplification (RPA), offers significant advantages for both instrumentation and assay development. RPA is tolerant to impure samples, amplifies DNA to detectable levels in as few as 5 minutes, and is available in a lyophilized form that can be transported to the point of care without requiring cold chain storage [7], [13], [14]. Lateral flow strips may be used for detection of amplified RPA products in low resource settings. In addition, RPA operates at a wide range of temperatures [7]. TwistDx recommends an incubation temperature of 37 degrees Celsius (the temperature of the human body) but notes that amplification may occur at temperatures as low as 25 degrees Celsius by using additional magnesium acetate, extending incubation time, and agitating reactions later in the incubation period [13]. Others have shown that even without adjusting the biochemistry of reactions, RPA retains reliable functionality between 31 and 43 degrees Celsius [15]. Although the possibility of incubating RPA reactions using body heat has been mentioned in previous work [5], [16], to the best of our knowledge, there are no examples of harnessing body heat to perform RPA in the literature.

In this paper, we explored the feasibility of using body heat to incubate RPA reactions for amplification of HIV-1 DNA. We chose this assay because detection of HIV-1 proviral DNA is an established method for early infant diagnosis [17], and the HIV-1 DNA RPA assay used here has been well-characterized elsewhere [18]. First we measured the temperature of mock reactions incubated at 4 body locations chosen to allow comfortable, convenient incubation. After demonstrating that the axilla is the ideal location for incubation, we investigated 3 commonly available materials to secure RPA reactions to the body. We also studied the effect of ambient conditions on incubation temperature to determine the ambient temperature range for which incubation with body heat may be feasible. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block to demonstrate that body heat may be harnessed to enable isothermal amplification of HIV-1 DNA.

To allow convenient incubation of RPA reactions under the arm, several methods were tested for securing tubes to the body. Mock reactions were secured by wrapping a 10 cm wide bandage ($12, CVS Pharmacy, USA), applying a 5 cm wide elastic sweat band ($1, Academy Sports and Outdoors, USA), and tying an 8 cm wide strip of cotton cloth (African chitenje fabric, approximately $4 per yard, outdoor market, Malawi) over the shoulder and under the arm (Figure 1). Volunteers incubated a tube containing 50 µL of water for 45 minutes using each method while the temperature was measured as previously described. These measurements were taken for five volunteers.

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. Figure 2 shows the temperature traces of mock RPA reactions incubated by 5 volunteers at 4 body locations. Mock reactions held in the axilla outside of clothing (Fig. 2A), taped to the abdomen under clothing (Fig. 2B), placed in a rear trouser pocket (Fig. 2C), and held in a closed fist (Fig. 2D) had average temperatures of 34.80.6, 31.31.7, 33.10.5, and 33.42.7 degrees Celsius, respectively. In less than three minutes, all mock reactions reached a temperature of 31 degrees Celsius, the temperature required for all RPA reactions to amplify DNA to detectable levels [15]. Because the temperature of mock reactions was closest to the temperature recommended for RPA (37 degrees Celsius) when incubated in the axilla, this site was chosen as the site of incubation for all following experiments.

Each plot shows the temperature traces of mock RPA reactions incubated by 5 volunteers at 1 of 4 body location tested. Mock reactions were (A) held in the axilla, (B) taped to the abdomen, (C) placed in a rear trouser pocket, and (D) held in a closed fist.

Several methods were tested for securing tubes to the body to allow convenient incubation of RPA reactions in the axilla. Figure 3 shows the temperature traces of mock RPA reactions incubated by 5 volunteers using 3 different methods. Mock reactions secured with a strip of cotton chitenje fabric (Fig. 3A), a bandage (Fig. 3B), and an elastic sweatband (Fig. 3C) reached an average temperature of 33.21.6, 32.91.2, and 33.50.7 degrees Celsius, respectively. The average time to reach 31 degrees Celsius using the chitenje fabric, a bandage, and an elastic sweatband was 2.02.7, 2.51.8, and 2.11.0 minutes, respectively. Because all methods produced similar temperatures, and cotton fabric is inexpensive and widely available in developing countries, tubes were secured with a strip of cotton fabric for all following experiments.

To demonstrate that body heat may be harnessed to incubate RPA reactions, RPA reactions were secured with a strip of cotton fabric and incubated in the axilla of ten volunteers in an office or laboratory at room temperature and in a cold room at 10 degrees Celsius, while control RPA reactions were incubated in a heat block. For experiments performed at room temperature, all reactions containing no HIV-1 DNA tested negative for both heating methods (Table 1). All reactions containing HIV-1 DNA tested positive when heated in a heat block, while 100% of 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. For experiments performed at 10 degrees Celsius, all reactions containing no HIV-1 DNA tested negative for both heating methods (Table 2). When heated in a heat block, all control reactions containing 100 copies of HIV-1 DNA tested positive, and 90% of reactions with 10 copies of HIV-1 DNA tested positive. The false negative sample may have been due to an experimental error. When samples were incubated with body heat, all reactions containing HIV-1 DNA tested positive. For both room temperature and cold room experiments, there was no significant difference between the signal-to-background ratio of the lateral flow strips for control reactions and reactions incubated with body heat when compared using a paired, one-tailed t-test.

We have demonstrated that RPA reactions may be incubated using body heat for amplification of HIV-1 DNA. Within a certain operating range, the temperature using this method is consistent over time and varies little from person to person. This method may be modified for convenience, as reactions may be incubated at several body locations and secured using available materials that may be reused for many experiments. The use of a plastic bag to contain reactions, while optional, may prevent contamination and provide a protective barrier between the user and the reaction components, which already pose little risk to the user. Temperature profiles and reaction results were not significantly affected by clothing material worn by the volunteer. Incubation of reactions using body heat is extremely inexpensive, as it obviates the need for any heating equipment, and the only consumables required are tubes and pipette tips. The method described here for incubating RPA reactions using body heat may be combined with any suitable DNA extraction method that is compatible with RPA. As RPA is tolerant to sample impurities, simple lysis methods such as boiling may adequately prepare samples for amplification [14]. In addition, this incubation method is compatible with other detection methods, including enclosed systems designed to reduce amplicon contamination, which may be more appropriate for point-of-care settings [22].

Finally, the sensitivity of DNA amplification was slightly lower when reactions were incubated with body heat at room temperature, as only 9 of 10 reactions with 100 copies of HIV-1 DNA tested positive. Notably, the reaction containing 100 copies of HIV-1 DNA that was classified as negative produced a faintly visible line at the test zone of the lateral flow strip (Fig. S1B); however, the SBR was slightly lower than the threshold for positive samples. To ensure that DNA is amplified to detectable levels and that results are clearly positive on lateral flow strips (Fig. S1), a longer incubation time may increase the SBR of lateral flow strip results, thereby increasing assay sensitivity. Once the incubation time is optimized, this method may serve as a low-cost, simple method for incubating RPA reactions in low resource settings.

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