In-situ fluorescent immunomagnetic multiplex detection of foodborne pathogens in very low numbers

Consumption of foods contaminated with pathogenic bacteria is a major public health concern. Foods contain microorganisms, the overwhelming majority of which are nonpathogenic, some are responsible for food spoilage, and some cause serious illness leading to death or a variety of diseases in humans. The key challenge in food safety is to rapidly screen foods to determine the presence of pathogens so that appropriate intervention protocols can be pursued. A simple fluorometric immunological method in combination with a magnetic concentration step was developed for rapid detection of target bacteria with high sensitivity and specificity in less than 2h without enumeration. The method constitutes performing an in-situ immunoassay on a magnetic bead through the formation of a sandwich complex of the target bacteria and the probe (detection antibody-denatured BSA labelled with fluorophores) followed by the release of fluorophores by means of enzymatic digestion with proteinase K. The limit of detection (LOD) was <5 CFU/mL of the tested pathogens (Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes) in buffer. When the pathogens were inoculated in foods (spinach, chicken, and milk), the LOD was under 5 CFU/mL for E. coli O157:H7, S. typhimurium and L. monocytogenes. Furthermore, the method was highly specific in detecting the target pathogens in a multiplex format. The developed in-situ fluorescent immunomagnetic sensor approach offers distinct advantages because it is rapid, highly sensitive, and easy to use and could therefore be potentially used as a pathogen screening tool.