Development and application of a molecular beacon-based recombinase-aided amplification method for multiplex detection of Staphylococcus aureus and aminoglycoside resistance genes

Staphylococcus aureus and its drug resistance in bovine mastitis pose a significant threat to raw milk biosafety. Three sets of primers and improved molecular beacon (IMB) probes were designed for fluorescence quantitative recombinase-aided amplification (IMB-RAA) method using the gyr B, aac (6′)- aph (2′′) and ant (4′)-Ia genes of S. aureus . The specificity of primers (IBMs) was shown species specificity for S . aureus , and the phenotypes were gentamicin and kanamycin resistance, respectively. The detection limits for positive plasmid DNA and artificially contaminated skim milk were 1.6 × 10 2 and 1.8 × 10 2 copies/μL, respectively, achieved within 20 min. The linear correlation coefficients for the three standard curves exceeded 0.99, with amplification efficiencies ranging between 90 % and 110 %. The triple detection system demonstrated strong repeatability. 160 milk samples were detected using the IMB-RAA method, with 33.13 % identified as S. aureus , achieving a consistency rate of 98.13 % with the GB4789.10–2016 standard. Gentamicin and kanamycin resistance genes were detected in 8 and 10 strains, respectively, with 100 % consistency with the observed antibiotic resistance phenotypes. Therefore, IMB-RAA offers a specific, sensitive, rapid, and convenient approach for the early diagnosis of bovine mastitis caused by S. aureus and for ensuring raw milk biosafety. • IMB-RAA fluorescent probe is an improved molecular beacon probe. • Triple IMB-RAA detection shows high specificity for IMBs and S. aureus gyr B primer. • aac (6′)- aph (2′′) and ant (4′)-Ia genotypes match GEN and KAN resistance phenotypes. • Triple IMB-RAA rapidly detects S. aureus and resistance genes in mastitis cow milk.