Recombinase aided amplification with photoreactive DNA-binding dye for rapid detection of viable Staphylococcus aureus

Abstract

Staphylococcus aureus is a common foodborne pathogenic microorganism. A modified propidium monoazide (PMAxx) dye combined with recombinase aided amplification (RAA) was proposed for the rapid and real-time detection of viable S. aureus. PMAxx, a kind of nucleic acid dye is used to distinguish viable bacteria from dead bacteria. Additionally, RAA technique has the advantage of a short detection time (20 min) and uniform temperature (39 °C), which is of great significance for the efficient detection of S. aureus. The S. aureus nuc gene was used as primer and probe in a microplate assay. The effect of PMAxx treatment exhibited permeation of the dead cell membranes that was observed under confocal laser scanning microscope (CLSM). After the enrichment of viable target bacteria ranging from 5.4 × 101 to 5.4 × 103 CFU/mL, the LOD for viable S. aureus was 102 CFU/mL under 3 h enrichment and 101 CFU/mL under 6 h enrichment in artificially contaminated milk, respectively. In the presence of nontarget bacteria at 106 CFU/mL, the system could detect 107 CFU/mL S. aureus in milk sample. The proposed PMAxx-RAA assay exhibited potential for the accurate and rapid viable S. aureus detection in milk.