Development and Application of Recombinase-Aided Amplification Combined with Lateral Flow Dipstick Assay for Rapid Detection of Staphylococcus aureus

In this study, our aim was to establish a sensitive and rapid approach for the visual and point-of-care detection of Staphylococcus aureus using recombinase-aided amplification (RAA) and lateral flow dipstick (LFD). We designed and screened three pairs of specific primers targeting conserved sequences in the nuc gene of S. aureus and optimized reaction conditions. The RAA-LFD assay took 20 min to amplify the target gene with a 200 nmol/L primer concentration at 33 ℃. The specificity analysis exhibited no cross-reaction with other pathogens. The sensitivity of the RAA-LFD method was as low as 4 fg/μL for DNA and 1.83 × 10² CFU/mL for pure cultures. The reliability was verified using artificially contaminated milk samples, showing that the limit of detection (LOD) was 1.83 × 10¹ CFU/mL after 6 h enrichment. When 64 milk samples were tested for S. aureus using the traditional culture method, polymerase chain reaction (PCR) and the RAA-LFD method, the results of RAA-LFD showed a high total coincidence rate of 95.3% and 98.4% with those of the two former methods respectively. The visual detection method established in this study has the characteristics of high specificity, high sensitivity, rapidity, simple operation, and low requirements for equipment, and can provide a new development direction for the detection of S. aureus in milk.