title: 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 <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.
authors.affiliation: Bindley Bioscience and Birck Nanotechnology Center, Department of Agricultural & Biological Engineering, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: Il-Hoon
authors.initials: IH
authors.lastname: Cho
authors.affiliation: Department of Food Science, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Mauer
authors.affiliation: Bindley Bioscience and Birck Nanotechnology Center, Department of Agricultural & Biological Engineering, Purdue University, West Lafayette, IN 47907, United States; Department of Food Science, Purdue University, West Lafayette, IN 47907, United States. Electronic address: josephi@purdue.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Irudayaraj
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D002645:Chickens; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005453:Fluorescence; D005506:Food Contamination / Q000032:analysis; D018189:Immunomagnetic Separation / Q000379:methods*; D057230:Limit of Detection; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D011200:Poultry / Q000382:microbiology*; D012486:Salmonella typhimurium / Q000302:isolation & purification*
keywords: Fluorescent detection; Foodborne pathogens; Highly sensitive; Magnetic separation; Rapid detection; Signal enhancement