LUMINESCENT METHODS TO DETECT VIABLE AND TOTAL ESCHERICHIA COLI O157:H7 IN GROUND BEEF^†

Abstract

Escherichia coli O157:H7 was inoculated into ground beef. Samples were incubated in E. coli broth plus novobiocin growth media at 37C for 5 h. Bacteria were captured by magnetic beads coated with anti‐E. coli O157:H7 antibodies and were treated with menadione, a membrane‐permeable electron‐transfer shuttering reagent, to oxidize the cellular nicotinamide adenine dinucleotide phosphate (NAD[P]H). Alternatively, bead‐captured bacteria were sandwiched by second anti‐E. coli O157:H7 antibodies labeled with peroxidase. The level of NAD(P)H and the enzyme activity of antibody‐bound peroxidase were then measured by luminol‐linked luminescence. The treatment of the E. coli O157:H7 with a bacterial protein extraction reagent diminished the luminescence associated with cellular NAD(P)H but not the luminescence associated with the sandwiched peroxidase label. Thus, the NAD(P)H‐linked luminescence, not the luminescence that was associated with the sandwiched complexes, was related to the viability of the E. coli O157:H7.

Detection of Salmonella Enteriditis from Egg Components Using Different Immunomagnetic Beads and Time-resolved Fluorescence

“…Dilutions of 1×10 8 cells/ mL were prepared in PBS and serially diluted to 1×10 4 cells/mL. The cells from each dilution were then treated with Bacterial Protein Extraction Reagent (B-PER; Pierce Technology, Rockford, IL, USA), a reagent that induced the loss of cell cytoplasmic content (Tu et al 2005), to stop any possible bacterial growth prior to the immunoassay.…”

Tunable Two-Dimensional Array Patterning of Antibody Annuli through Microsphere Templating

“…5,6 Functional antibody arrays are often used as the binding structure for the detection of specific biomolecules and larger objects such as viruses and bacteria, 7 with high-density sandwich assays being extensively studied and utilized. 8,9 The immobilization of antibodies on solid supports in a patterned fashion is an essential step in immunoanalysis and a prerequisite for a number of immunoassay formats. 11 The techniques used to pattern protein microarrays can be divided into lithographic techniques, 2,10,12 microspot printing, 2,[13][14][15][16] and methods exploiting chemical heterogeneity on surfaces.…”

“…The specific adsorption of proteins from complex analytes such as serum, total cell extracts, and urine at solid−liquid interfaces constitutes an essential element in the fields of proteomics, where microarray technologies are often utilized to enable the evaluation of tens of thousands of molecular interactions simultaneously . Here the size as well as the density of the binding structure is essential for ensuring that the high-throughput analysis has adequate specificity and sensitivity. , Also, because proteins, in contrast to nucleic acids, cannot yet be replicated, the development of protein microarrays and nanoarrays for analyzing protein expression and functions of complex protein mixtures remains a challenging and active research area. , Functional antibody arrays are often used as the binding structure for the detection of specific biomolecules and larger objects such as viruses and bacteria, with high-density sandwich assays being extensively studied and utilized. , The immobilization of antibodies on solid supports in a patterned fashion is an essential step in immunoanalysis and a prerequisite for a number of immunoassay formats…”

DETECTION OF SALMONELLA ENTERITIDIS IN SHELL EGG CONTENTS BY IMMUNOMAGNETIC CAPTURE AND TIME‐RESOLVED FLUORESCENCE1

“…In the process of searching for better methodologies for pathogen detection in foods, we have incorporated the use of immunomagnetic beads (IMBs) for the capture and concentration of Escherichia coli O157:H7 with various biosensor signaling methods including digital fluorescent imaging of intact cells (Tu et al. 1998), metabolically regulated adenosine triphosphate and nicotanamide adenine dinucleotide, reduced form, bioluminescence of viable cells (Tu et al. 1999, 2005) and light addressable potentiometric sensing for general applications (Tu et al.…”