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

Abstract

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 gyrB, 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 × 10² and 1.8 × 10² 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.