Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in meat using multiplex immunomagnetic separation and multiplex real-time PCR

This study aimed to develop a combination of immunomagnetic separation (IMS) and real-time PCR (qPCR) techniques to detect and analyze Salmonella spp. (SAL), Listeria monocytogenes (LM), and Escherichia coli O157:H7 (O157: H7) in meat samples quickly and accurately. Bacteria-specific immunomagnetic beads (IMBs) were prepared from carboxyl magnetic beads and affinity-purified polyclonal antibodies using a novel magnetic bead activator. Multiplex immunomagnetic separation (mIMS) and multiplex qPCR were performed to detect the three pathogenic bacteria. Both IMS and mIMS showed a high capture efficiency with good repeatability. The standard curve of the three pathogenic bacteria was linear within a concentration range of 10 2 –10 8 CFU/mL. Without bacterial enrichment, the detection limits for SAL, LM, and O157:H7 were 1.2 × 10 3 , 3.3 × 10 3 , and 1.1 × 10 3 CFU per 25 g, respectively. Following a 5-h enrichment period, the corresponding detection limits increased to 12, 33, and 11 CFU per 25 g, respectively. Evaluation of 160 meat samples revealed that the sensitivity, specificity, and accuracy of the method were 100%, 98.1%, and 98.5%, respectively. Collectively, the IMS-based triplex qPCR method developed in this study improves detection sensitivity with reduced detection time and, therefore, has broad prospects in detecting pathogenic bacteria in food.