title: A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.
abstract: Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform.
authors.affiliation: Center of Nanotechnology, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, USA. Electronic address: baskarmicrobiologist@gmail.com.
authors.forename: Baskar
authors.initials: B
authors.lastname: Balakrishnan
authors.affiliation: Center of Nanotechnology, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, USA.
authors.forename: Syed
authors.initials: S
authors.lastname: Barizuddin
authors.affiliation: Small Animal Research Facility, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, USA.
authors.forename: Tumen
authors.initials: T
authors.lastname: Wuliji
authors.affiliation: Center of Nanotechnology, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, USA. Electronic address: DweikM@LincolnU.edu.
authors.forename: Majed
authors.initials: M
authors.lastname: El-Dweik
mesh_terms: D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique; D005516:Food Microbiology; D006801:Humans; D008460:Meat / Q000382:microbiology*; D012680:Sensitivity and Specificity
keywords: E. coli O157:H7; Food safety; Immunofluorescence; Magnetic bead; Protein-A; TRITC