Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA

Abstract

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Figure 1. The prototype SAT heater used to incubate RPA reactions.

(A) A photograph of an activated SAT heater, the reaction tube is filled with blue dye to improve definition. (B). A cross sectional diagram depicting the SAT heater and other test components.

Figure 2. Median peak temperatures produced by different concentrations of SAT when held and activated under a variety of ambient temperatures.

Ambient temperatures of SAT mixtures during activation: blue 10°C; green 15°C; orange 20°C; turquoise 25°C and red 30°C; SAT, Sodium acetate trihydrate.

Figure 3. Maps depicting sub Saharan Africa and the frequency (fraction of days) on which mean afternoon (12–18 hr local time) surface air temperatures are below 43°C during the warm season (A), exceed 30°C in the warm season (B) and are above 10°C in the cold season (C).