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

Area of Science:

• Molecular Biology
• Biotechnology
• Food Safety

Background:

• Fluorescent probe-based recombinase aided amplification (RAA) is rapid but requires complex probe design, limiting its use.
• Developing simpler, more sensitive detection methods for foodborne pathogens is crucial for public health.

Purpose of the Study:

• To develop a novel EvaGreen dye-based RAA (EvaGreen-RAA) assay for detecting Pseudomonas fluorescens (PF) and Bacillus cereus (BC) in milk.
• To evaluate the sensitivity and simplicity of EvaGreen-RAA compared to conventional RAA.
• To investigate a dual EvaGreen-RAA assay for simultaneous detection of mixed BC and PF.

Main Methods:

• A novel EvaGreen dye-based RAA (EvaGreen-RAA) assay was developed using self-avoiding molecular recognition system (SAMRS) primers.
• Conventional RAA served as the reference method.
• Sensitivity was assessed using nucleic acids from recombinant plasmids and simulated milk specimens, including a dual-pathogen assay.

Main Results:

• EvaGreen-RAA showed superior sensitivity over conventional RAA, with detection limits of 1 copy/µL versus 10 copies/µL for BC and PF plasmids.
• In simulated milk, EvaGreen-RAA detected BC at 100 CFU/mL and PF at 200 CFU/mL, significantly lower than conventional RAA (400 CFU/mL and 600 CFU/mL).
• The dual EvaGreen-RAA assay successfully detected mixed BC and PF at 200 CFU/mL each.

Conclusions:

• EvaGreen-RAA offers enhanced simplicity and sensitivity compared to fluorescent probe-based RAA.
• This assay provides a novel approach for developing multiplex pathogen detection systems using melting curve analysis.
• The EvaGreen-RAA assay is a promising tool for rapid and sensitive detection of foodborne pathogens in milk.