Development of A Novel EvaGreen-Dye Based Recombinase Aided Amplification Assay Using Self-Avoiding Molecular Recognition System Primers

Fluorescent probe-based recombinase aided amplification (RAA) offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design, restricting its widespread application.

A novel EvaGreen dye-based RAA (EvaGreen-RAA) assay utilizing self-avoiding molecular recognition system (SAMRS) primers was developed for the detection of Pseudomonas fluorescens (PF) and Bacillus cereus (BC) in milk. Conventional RAA was used as a reference method. Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens. Additionally, a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.

The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA, with detection limits of 1 copy/µL versus 10 copies/µL for both BC and PF plasmids, respectively. In simulated milk specimens, EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL, respectively, compared to 400 CFU/mL and 600 CFU/mL for conventional RAA. The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.

The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA, offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.