title: Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.
abstract: Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead-based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.
authors.affiliation: Department of Molecular Biosciences, Arkansas State University, Jonesboro, Arkansas 72401, USA.
authors.forename: Muhsin
authors.initials: M
authors.lastname: Aydin
authors.affiliation: Department of Biological Sciences, Arkansas State University, Jonesboro, Arkansas 72401, USA.
authors.forename: Gene P D
authors.initials: GP
authors.lastname: Herzig
authors.affiliation: Emerging Pathogens Institute and Department of Animal Sciences, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Kwang Cheol
authors.initials: KC
authors.lastname: Jeong
authors.affiliation: Department of Biological Sciences, Arkansas State University, Jonesboro, Arkansas 72401, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Dunigan
authors.affiliation: Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, New Jersey 07102, USA.
authors.forename: Parth
authors.initials: P
authors.lastname: Shah
authors.affiliation: Department of Food Science and Human Nutrition, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, USA.
authors.forename: Soohyoun
authors.initials: S
authors.lastname: Ahn
mesh_terms: D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial / Q000379:methods; D003257:Consumer Product Safety; D004797:Enzyme-Linked Immunosorbent Assay; D019453:Escherichia coli O157 / Q000276:immunology / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000737:chemistry; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D005517:Foodborne Diseases; D006801:Humans; D007118:Immunoassay / Q000379:methods; D018189:Immunomagnetic Separation / Q000379:methods* / Q000592:standards*; D008461:Meat Products / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014439:Tyramine / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D014439:Tyramine