DNA Detection Using Recombination Proteins

Abstract

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.

Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification

“…3 Recombinase polymerase amplification (RPA) offers significant advantages over other isothermal amplification techniques for point-of-care applications: it requires a lower amplification temperature, is tolerant to impure samples, amplifies targets to detectable levels within minutes, and uses lyophilized enzymes to enable storage and transport at room temperature. 4,5 For these reasons, a number of recent reports have proposed RPA-based strategies for the detection of pathogens. [6][7][8][9][10] Although some papers demonstrate a relationship between nucleic acid concentration and onset of amplification, 10,11 to the best of our knowledge, RPA has not yet been implemented to quantify sample concentration using a standard curve.…”

Novel tools for the surveillance and control of dengue: findings by the DengueTools research consortium

“…We developed a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay that is quick and can be undertaken in low resource settings [15]. The RPA is an isothermal nucleic acid amplification technology where reactions take place at a low and constant temperature between 22°C and 45°C [16]. Fluorescence was detected using a portable real-time fluorometer, ‘Twista®’, which has a small footprint and can be rendered entirely portable with the addition of a rechargeable battery pack, developed by the private sector collaborator, TwistDx.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Shin et al [69] have demonstrated one of the most sophisticated and sensitive approaches for isothermal PCR-like amplification of ds-DNA using a solid-phase immobilized primer. This was carried out using recombinase polymerase amplification (RPA) [70,71] which employs a uvsX recombinase enzyme of specific activity isolated from T4 phage. In this case the enzyme is intended to "substitute" the thermal denaturation step normally performed during PCR amplification.…”