Slipchip-based immunomagnetic separation combined with loop-mediated isothermal amplification for rapid detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk

Abstract

Rapid detection of antibiotic resistance genes (ARGs) of food microorganisms is critical for preventing the spread of ARGs via the food chain. In this study, slipchip-based immunomagnetic separation (IMS) combined with loop-mediated isothermal amplification (LAMP) was developed for rapid, specific, sensitive and multi-sample detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk. First, the magnetic nanoparticles coated with anti-B. cereus polyclonal antibodies were mixed with samples in the magnetic capture chamber of the slipchip, and the capture efficiency of Bacillus cereus (105 CFU/mL) can reach 80% after incubation for 1h. Then, the captured bacteria were boiled in the lysis chamber using a heating block and the extracted DNA was added into the LAMP reaction as templates. Finally, real-time fluorescent LAMP detection for tetL gene of Bacillus cereus was performed at 65 °C for 50 min. Under optimal conditions, the IMS-LAMP could detect 11.6 CFU/mL of Bacillus cereus with tetL gene in pure culture. Furthermore, the limit of detection of Bacillus cereus with tetL gene in pasteurized milk was 21.5 CFU/mL by the slipchip-based IMS-LAMP within 2 h. Therefore, this method is potentially useful for the rapid detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk and could be extended for other resistance genes.