Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification

Abstract

The recombinase polymerase amplification (RPA)-based isothermal nucleic acid amplification strategy is one of the most rapidly developing methods for performing nucleic acid tests outside laboratory settings. However, the reaction mechanism of RPA results in unavoidable concerns related to nonspecific and off-target binding of the recombinase enzyme that lead to false-positive results, and thus successful RPA signal detection outside the laboratory has yet to be demonstrated. Here, we developed one-inch gel electrophoresis for simple and portable electrophoretic analysis of RPA products to discriminate true-positive results from false-positive and/or negative results generated during the RPA reaction and described a protocol for field-amenable direct sampling and RPA assay using Whatman FTA (Flinders Technology Associates) cards and body heat. First, we optimized a near-to-patient RPA protocol for the detection of Leishmania species using 18S rRNA gene analysis. Next, we introduced a handheld electrophoretic device using our 1-inch polyacrylamide gel system to perform a 5 minute electrophoretic analysis of RPA results. Our results demonstrate that the combination of robust RPA with FTA card-based direct sampling tools and portable electrophoretic devices can revolutionize nucleic acid-based molecular diagnostics for people in settings with poor healthcare infrastructure.