a b s t r a c tRecombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37e42 C, with minimal sample preparation and capable of amplifying as low as 1e10 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.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10−11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).