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

Abstract

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 <5CFU/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 5CFU/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.