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.

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

“…One advantage is that the RT-exoRPA does not require the reaction temperature to be precisely controlled. Furthermore, although in this study the amplification signal of the RT-exoRPA was analysed using a laboratory real-time PCR instrument, in principle only a simple heating device is needed to achieve a temperature around 37 • C (Craw and Balachandran, 2012;Lillis et al, 2014). This simple heating instrument is a major advantage of the RPA assay in places experiencing power failures as the reaction could proceed although at a slower pace.…”

Rapid Detection of Salmonella Enterica Serovar Enteritidis from Eggs and Chicken Meat by Real‐Time Recombinase Polymerase Amplification in Comparison with the Two‐Step Real‐Time PCR

“…This assay allows the use of common equipment, like a simple water bath, due to the relatively low and flexible reaction temperature (Crannell et al 2014;Lillis et al 2014). Our results show that the real-time RPA is considerably faster than the fast detection mode of the two-step real-time PCR and LAMP assays, as well as there are no losses in sensitivity and specificity in comparison with other diagnostic systems.…”

“…This is because RPA operates on an enzyme-derived primer binding process rather than by physio-chemical hybridization of primers (Lillis et al 2014). In previous studies, RPA had been demonstrated using a simple noninstrumented heat source or just human body heat (Crannell et al 2014;Lillis et al 2014). In our study, a real-time PCR thermal cycler, which is a very common instrument in the area of life science, was used for developing the real-time RPA.…”

“…This is unique when compared with other isothermal amplification assays (Piepenburg et al 2006;Zanoli and Spoto 2013). Moreover, RPA is tolerant to temperature fluctuation and does not require a precisely controlled temperature during the reaction (Crannell et al 2014;Lillis et al 2014). This is because RPA operates on an enzyme-derived primer binding process rather than by physio-chemical hybridization of primers (Lillis et al 2014).…”

“…Moreover, RPA is tolerant to temperature fluctuation and does not require a precisely controlled temperature during the reaction (Crannell et al 2014;Lillis et al 2014). This is because RPA operates on an enzyme-derived primer binding process rather than by physio-chemical hybridization of primers (Lillis et al 2014). In previous studies, RPA had been demonstrated using a simple noninstrumented heat source or just human body heat (Crannell et al 2014;Lillis et al 2014).…”