Establishment of a real-time fluorescence and visual colorimetric detection method for Staphylococcus aureus based on loop-mediated isothermal amplification

Abstract

A loop-mediated isothermal amplification (LAMP) method combined with calcein was established for the detection of Staphylococcus aureus. LAMP primers were designed targeting the ASL18_04625 species-specific gene screened through bioinformatics and PCR analysis. The target gene and detection method demonstrated 100% specificity for S. aureus and no cross-reactions with other pathogens. For LAMP reactions, the results could be directly observed with the naked eye or evaluated by S-shaped amplification curves of fluorescence signals. The assay's detection limit was 3.395 × 10⁻⁴ ng/μL genomic DNA or 3.21 CFU/mL pure bacterial culture. Furthermore, the target bacteria of 8.5 × 10⁵–8.5 × 10⁰ CFU/mL could be accurately detected in spiked milk samples, and the overall detection process could be completed within 40 min. This method improved the accuracy and convenience for S. aureus detection and provied a dependable tool for the rapid screening of S. aureus.