Recombinase polymerase amplification: Basics, applications and recent advances

Abstract

Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37-42°C, with minimal sample preparation and capable of amplifying as low as 1-10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.

Keywords: Isothermal amplification; Multiplexing; Recombinase polymerase amplification; Solid-phase amplification.

Fig. 1

RPA amplification scheme. Recombinase proteins form complexes with each primer (A), which scans DNA for homologous sequences (B). The primers are then inserted at the cognate site by the strand-displacement activity of the recombinase (C) and single stranded binding proteins stabilise the displaced DNA chain (D). The recombinase then disassembles leaving the 3′-end of the primers accessible to a strand displacing DNA polymerase (E), which elongates the primer (F). Exponential amplification is achieved by cyclic repetition of this process.

Fig. 2

Nfo probe and lateral flow strip. The Nfo probe is exchanged at the cognate site by recombinase proteins and nfo nuclease cleaves the probe on the THF residue. The blocking group is released and the probe is thus converted into a primer. The double labelled amplicon obtained from amplification is mixed with a dye and loaded onto the sample pad of a lateral flow strip (A). The dye binds to the amplicon in the mixture pad (B) and the dye-amplicon complex is captured by antibodies immobilised on the detection line (C). The excess of dye is captured by antibodies in the control line (D).

Fig. 3

Schematic representations of biosensors/POC devices using RPA: (i) Bridge flocculation assay ; (ii) a disposable plastic and paper device (64); (iii) lab in a suitcase ; (iv) multiplexed lateral flow assay [57)]; (v) solid phase amplification on DVDs ; (vi) electrochemical solid phase amplification (Figures modified from original publications cited).

Fig. 4

Exo probe and Nfo probe for fluorescent real-time detection.