Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation.

Abstract

The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10min and visualization of the amplicons through LF strips for 5min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40CFU per reaction. The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.