Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7 1 Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. 1 (original) (raw)
Related papers
Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157:H7
Journal of Immunological Methods, 1998
A new approach for rapid assay of bacteria in liquid samples is described. Cells were labeled by incubation with an enzyme-antibody conjugate and captured by filtration of the samplerconjugate mixture through a 0.2 mm filter. The enzyme-labeled cells were detected by placing the filter on the surface of an electrode, incubating with enzyme substrate, and measuring the current produced by oxidation of the electroactive enzyme product. Assay time was 25 min and a detection limit of ; 5000 cellsrml was obtained for E. coli O157:H7. Background current due to non-specific binding of conjugate to the filter was the primary factor controlling the detection limit, and fewer than 50 cells could be detected when Ž. very small sample volumes 10 ml were used to minimize background current. q 1998 Elsevier Science B.V.
2003
Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluoresceintagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 mg antibody/1 million ...
Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7
Biosensors and Bioelectronics, 1999
A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.
…, 2003
As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluoresceintagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 mg antibody/1 million bacteria, and capillaries dynamically coated with poly-N-hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6-8 h, which is less than that realized in most commercial assays presently available.
Journal of Microbiological Methods, 2003
We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10 4 cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10 4 E. coli O157:H7 cells were V 10% in the presence of ca. 10 8 K. pneumoniae, K. oxytoca, or E. cloacae cells. None of these strains gave a positive IM-ECL signal. Although competitive binding decreased sensitivity, there still was a linear correlation between ECL signal and higher E. coli O157:H7 cell concentrations. These studies indicate that IM-ECL in conjunction with MLB enrichment is capable of quantitatively detecting as few as 10 3 to 10 5 E. coli O157:H7 cells ml À 1 , depending on percent recoveries, in enriched samples that contain ca. 10 9 total lactose-fermenting bacteria ml À 1. Assuming comparable growth rates for E. coli O157:H7 and other lactose-fermenting bacteria in MLB, it may be possible to detect as few as one E. coli O157:H7 in 100 ml of raw water containing as many as 10 4 to 10 6 lactose-fermenting bacteria (i.e., total coliforms).
Applied and Environmental Microbiology, 1988
An 0-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli 0157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli 0157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli 0157:H7, the organism was isolated at levels of up to 103/g. The lower limit of sensitivity was 10 E. coli 0157 per g of meat. Specific typing for E. coli 0157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.
An amperometric culture-based method was developed for rapid detection of viable Escherichia coli in water. The bacteria were recovered by filtration and incubated in a selective medium, lauryl sulphate broth (LSB) supplemented with the substrate 4-aminophenyl--d-galactopyranoside (4-APGal) at 44.5 • C. The electrochemically active molecule 4-aminophenol (4-AP) was produced after hydrolysis of 4-APGal by the enzyme -galactosidase. 4-AP was measured by amperometry and was detected at a due concentration of E. coli. The time necessary for reaching that concentration was inversely related to the initial E. coli concentration of the sample. Environmental samples and suspensions of E coli IT1 were assayed. 4-AP was detected after 7.3 and 2.0 h in samples containing initial concentrations of E. coli IT1 of 4.5 and 4.5×10 6 cfu ml −1 , respectively. For environmental samples with initial E. coli concentrations of 1.0 and 2.0×10 3 cfu ml −1 , 4-AP were detected after 10 and 6.6 h, respectively.
Clinical Diagnostic Laboratory Immunology, 1998
E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocki...
Rapid Detection ofEscherichia coliO157:H7 in Water
Journal - American Water Works Association, 2007
Escherichia coli O157 can cause life-threatening disease in the young and the elderly, and numerous outbreaks have been reported. Biochemical detection procedures are inappropriate for environmental monitoring, and simple, rapid, and sensitive isolation and detection procedures are required for improved surveillance. Four immunological assays (Reveal®, Eclipse TM , ImmunoCard STAT!, and the E. coli O157 Antigen Detection Test) and one molecular assay (BAX®) were evaluated to measure sensitivity for E. coli O157:H7 detection in water. Promising procedures were combined with sample concentration, enrichment, and capture using immunomagnetic separation. Method sensitivity was determined using various environmental water matrixes in both single-and multilaboratory evaluations to assess method robustness. Overall method sensitivity was ±0.05 cfu/mL using detection by both Reveal and BAX methods, which was considered suitable for environmental screening. The BAX method, because of lower cross-reactivity, was effective for both raw and finished water analyses whereas the Reveal method was suited for finished water. BY ZIA BUKHARI, JANICE R. WEIHE, AND MARK W. LECHEVALLIER scherichia coli O157:H7 has become recognized as a significant human pathogen and can lead to hemorrhagic colitis, hemolytic uremic syndrome (HUS), or thrombotic thrombocytopenic purpura, with serious cases of disease being fatal, especially in children and elderly populations. The Centers for Disease Control and Prevention estimates the annual disease burden of E. coli O157:H7 at approximately 40,000, with up to 250 deaths and a financial cost of $250-350 million (Boyce et al, 1995); however, the true incidence may be underestimated. Some estimates have suggested up to 73,000 cases annually, with 11,000 occurring from waterborne transmission (Shelton & Karns, 2001). Epidemiologic investigations of outbreaks suggest a low infectious dose for E. coli O157:H7 (i.e., 10-200 organisms; Willshaw et al, 1994) and, irrespective of whether the outbreak is foodborne (contaminated ground beef) or waterborne (untreated water supplies contaminated with animal feces), cattle are considered an important species for harboring E. coli. The zoonotic effect of E. coli
Rapid detection of Escherichia coli in water by a culture-based amperometric method
Analytica Chimica Acta, 2001
An amperometric culture-based method was developed for rapid detection of viable Escherichia coli in water. The bacteria were recovered by filtration and incubated in a selective medium, lauryl sulphate broth (LSB) supplemented with the substrate 4-aminophenyl--d-galactopyranoside (4-APGal) at 44.5 • C. The electrochemically active molecule 4-aminophenol (4-AP) was produced after hydrolysis of 4-APGal by the enzyme -galactosidase. 4-AP was measured by amperometry and was detected at a due concentration of E. coli. The time necessary for reaching that concentration was inversely related to the initial E. coli concentration of the sample. Environmental samples and suspensions of E coli IT1 were assayed. 4-AP was detected after 7.3 and 2.0 h in samples containing initial concentrations of E. coli IT1 of 4.5 and 4.5×10 6 cfu ml −1 , respectively. For environmental samples with initial E. coli concentrations of 1.0 and 2.0×10 3 cfu ml −1 , 4-AP were detected after 10 and 6.6 h, respectively.