Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics

Abstract

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

Recombinase polymerase amplification: Basics, applications and recent advances

“…In a more challenging approach, termed bridge amplification, both forward and reverse primers are immobilised on a surface. However, the vast majority of reports describing RPA exploit solution-phase amplification [25,26]. In solution-phase, due to the unimpeded diffusion of primers and reaction reagents, amplification kinetics are favoured and the achieved limit of detection is subsequently usually better and amplification is achieved in a faster time than solid-phase.…”

A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3

“…However, this method requires thermal cycling equipment and the settings of professional diagnostic laboratory. Moreover, most nucleic acid amplification technologies assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation [8]. With a reaction time of 1-2 h, these can also be fairly time-consuming.…”

“…The commercial reverse transcriptase kits (PrimeScript RT Master Mix; TaKaRa, Dalian, China) incubate the samples for 15 min at 37°C. The RPA tolerates temperatures ranging from 30 to 42°C without losing reaction efficiency and has been so far already successfully used to construct rapid detection of https://doi.org/10.1016/j.mcp.2018.08.004 Received 10 July 2018; Received in revised form 15 August 2018; Accepted 19 August 2018 many pathogens [8,[10][11][12][13].…”

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection

“…A novel isothermal detection of recombinase polymerase amplification (RPA) assay has been used for the diagnosis of many pathogens in recent years [5]. In this test, recombinase uvaX, DNA polymerase Bsu, single-strand binding protein (SSBP) gp32, and specific oligonucleotide primers were used, and the reaction started with magnesium acetate.…”