DNA Fragment Fusion and Nucleic Acid Detection by Fusion Recombinase-Aided Amplification

Abstract

Constructing fusion DNA fragments is frequently used for genetic engineering purposes. To date, fusion PCR is one of the most popular approaches for generating fusion DNA fragments. Here, we describe a novel method for DNA fusion based on the isothermal DNA amplification technique, recombinase-aided amplification (RAA). We demonstrate that this method, termed “fusion RAA”, can assemble two to three DNA fragments to generate a fusion fragment of up to ∼1 kb in a one-pot reaction within 40 min at 37 °C. We further demonstrate that fusion RAA can realize fragment insertion, deletion, and base mutation. Moreover, we show that fusion RAA can be harnessed to facilitate pathogen detection by simultaneously targeting two genes in one RAA assay, as demonstrated by the rapid and simplified detection of methicillin-resistant Staphylococcus aureus (MRSA). Based on fusion RAA, we establish two novel pathogen detection platforms, FREAC (Fusion REcombinase-aided Amplification combined with CRISPR/Cas13a) and FREAL (Fusion REcombinase-aided Amplification combined with Lateral flow assay). Using these two platforms, we can detect clinical MRSA strains within 55 min with high specificity and a limit of detection of 150 copies/μL of genomic DNA, highlighting their potential as user-friendly platforms for nucleic acid detection.