Triplex immunomagnetic separation combined with loop-mediated isothermal amplification on a microfluidic chip for simultaneous visual detection of Escherichia coli O157:H7, Salmonella enteritidis and Staphylococcus aureus in milk

Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus are widely present in food and can cause serious and large-scale cases of illness worldwide every year. Herein, we developed a triplex immunomagnetic separation (IMS) combined with loop-mediated isothermal amplification (LAMP) on a microfluidic chip (triplex IMS-mLAMP) for the simultaneous detection of these three important foodborne pathogenic bacteria. To optimize the triplex IMS method, the CEs of the three types of IMBs were determined for different particle sizes, amounts and incubation times. Under optimal reaction conditions, the CEs for the three bacteria were between 65.4% to 71.1% in PBS, and between 41.1% to 47.5% in milk, respectively. After triplex IMS, DNA from target bacteria captured by magnetic beads was extracted and subjected to a multiplexed LAMP reaction in a centrifugal microfluidic chip that allowed for the simultaneous detection of up to 8 individual milk samples. The results were judged directly by the color change. The method only detected the three target bacteria and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of the triplex IMS-mLAMP as low as 1.2 × 102 CFU/mL for E. coli O157:H7 and S. enteritidis, and 2.5 × 102 CFU/mL for S. aureus in milk samples. The overall testing time of the triplex IMS-mLAMP was just 80 min. This is the first report that IMS-mLAMP was used for simultaneous capture and detection of E. coli O157:H7, S. enteritidis, and S. aureus in milk. This cost-effective, easy-to-use, no need for expensive laboratory-based equipment, integrated on-chip colorimetric multi-target detection system opens a novel pathway for rapid diagnostic screening of pathogens in food.