AbstractBackgroundFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.