B subunit of cholera toxin produced in Escherichia coli

An engineered E. coli strain containing high expression level of CT-B subunits has been obtained by the application of recombinant DNA techniques. The B subunit can be secreted into the medium and reaches 20-40 micrograms/ml when this strain is incubated in a 50 l fermentation tank. The CT-B subunit purified with affinity chromatography in E. coli has the same characters as the natural CT-B subunit in molecular weight, N terminal amino acid analysis and antigenicity. The CT-B subunit has good immunogenicity and can be used as a preparation for protecting against diarrhea caused by V. cholera and enterotoxigenic E. coli. It can also be used as a vector for hepatins.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.     

Aggregation behavior and size of lipopolysaccharide from Escherichia coli O55:B5

Dynamic light scattering, steady-state fluorescence, NMR diffusometry and cryo-TEM have been used to gain more insight into the aggregation behaviour of LPS from Escherichia coli O55:B5. Knowledge of this behaviour of the amphiphilic LPS molecule is in many cases of importance for the design of experiments and interpretation of results when using LPS in solution. The aim of this work was to study the aggregation and determine the aggregate size of E. coli O55:B5. The mean hydrodynamic radius of the LPS aggregates was determined by NMR diffusometry and dynamic light scattering to 14 and 26nm, respectively. The cryo-TEM technique revealed predominately spherical aggregates of 9-19nm. We wish to report 10mug/ml as the aggregation start for LPS E. coli O55:B5 in PBS buffer, pH 7.2. We suggest that the aggregation is a continuous process that starts at 10mug/ml and proceeds up to 300mug/ml.     

Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains.     

An anti E. coli O157:H7 antibody-immobilized microcantilever for the detection of Escherichia coli (E. coli).

A silicon microcantilever sensor was developed for the detection of Escherichia coli O157:H7. The microcantilever was modified by anti-E. coli O157:H7 antibodies on the silicon surface of the cantilever. When the aquaria E. coli O157:H7 positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the E. coli O157:H7 antigen by the antibodies on the surface of the microcantilever. A negative control sample that does not contain E. coli O157:H7 antigen did not cause any bending of the microcantilever. The detection limit of the sensor was 1 x 10(6) cfu/mL when the assay time was < 2 h.     

DuPont qualicon BAX system real-time PCR assay for Escherichia coli O157:H7

Evaluations were conducted to test the performance of the BAX System Real-Time PCR assay, which was certified as Performance Tested Method 031002 for screening E. coli O157:H7 in ground beef, beef trim, spinach, and lettuce. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the FDA-BAM and the USDA-FSIS culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined affect the performance of the assay. An accelerated shelf life study determined an initial 36 month shelf life for the test kit.     

Detex for detection of Escherichia coli O157 in raw ground beef and raw ground poultry.

A method for detection of Escherichia coli O157 in beef and poultry is presented. The method is antibody-based and uses a patented antibody-specific metal-plating procedure for the detection of E. coli O157 in enriched meat samples. Both raw ground beef and raw ground poultry were tested as matrixes for the organism. The sensitivity and specificity of the assay were 98 and 90%, respectively. The accuracy of the assay was 96%. Overall, the method agreement between the E. coli O157 Detex assay and the U.S. Department of Agriculture/Food Safety Inspection Service method was 96%. Sample temperature upon loading of the apparatus was critical to the observed false-positive rate of the system.     

Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181

Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The ¹H and ¹³C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR ¹³C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.     

Chemiluminescent enzyme-linked immunosorbent assay on a strip to detect Escherichia coli O157:H7

A fast and sensitive chemiluminescent enzyme-linked immunosorbent assay method to measure pathogenic bacteria, Escherichia coli O157:H7, on immuno-chromatographic membrane was studied. Non-specific binding of proteins on membrane strip was controlled to attain the best performance of immunosensor by optimising the composition of a running buffer. The specificity of the proposed immunostrip was confirmed by conducting experiments for four different micro-organisms. A chemiluminescent signal could be successfully generated from a proposed immunostrip sensing system, and a significant change in the chemiluminescent light intensity with the concentration of target microbes was obtained. E. coli O157:H7 could be quantitatively measured in the range of 1.1?×?10^3?–1.1?×?10⁷ CFU (colony forming units) mL^?1 within 16?min by using the developed chemiluminescent immunostrip.     

Structural analysis of lipid A from Escherichia coli O157:H7:K- using thin-layer chromatography and ion-trap mass spectrometry

Rapid separation and structural identification of lipid A from Escherichia coli were performed using thin-layer chromatography (TLC) and mass spectrometry (MS). After the resolved spot of the lipid A had been scraped from TLC plate, the sample was re-extracted from the removed powder with chloroform-methanol (2 : 1, v/v) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) ion-trap MS. For detailed structural characterization, multiple-stage mass analysis (MS(4)) of the major species in ESI-MS/MS provided important information about the series of fragment ions. The dominant fragment ions in each MS stage were produced from the loss of fatty acyl groups mainly driven by charge-remote processes, and this information about the fragment ions can be used to deduce the composition or the position of the fatty acid substituent in the lipid A. In contrast, MALDI-TOFMS indicated that fragmentation resulted from charge-driven processes. Molecular mass profiling and fragmentation analysis provides essential information for clarifying the detailed structure of the lipid A from E. coli O157:H7:K(-).     

Double interdigitated array microelectrode-based impedance biosensor for detection of viable Escherichia coli O157:H7 in growth medium

Double interdigitated array microelectrodes (IAM)-based flow cell was developed for an impedance biosensor to detect viable Escherichia coli O157:H7 cells after enrichment in a growth medium. This study was aimed at the design of a simple flow cell with embedded IAM which does not require complex microfabrication techniques and can be used repeatedly with a simple assembly/disassembly step. The flow cell was also unique in having two IAM chips on both top and bottom surfaces of the flow cell, which enhances the sensitivity of the impedance measurement. E. coli O157:H7 cells were grown in a low conductivity yeast-peptone-lactose-TMAO (YPLT) medium outside the flow cell. After bacterial growth, impedance was measured inside the flow cell. Equivalent circuit analysis indicated that the impedance change caused by bacterial growth was due to double layer capacitance and bulk medium resistance. Both parameters were a function of ionic concentration in the medium, which increased during bacterial growth due to the conversion of weakly charged substances present in the medium into highly charged ions. The impedance biosensor successfully detected E. coli O157:H7 in a range from 8.0 to 8.2x10(8)CFUmL(-1) after an enrichment growth of 14.7 and 0.8h, respectively. A logarithmic linear relationship between detection time (T(D)) in h and initial cell concentration (N(0)) in CFUmL(-1) was T(D)=-1.73logN(0)+14.62, with R(2)=0.93. Double IAM-based flow cell was more sensitive than single IAM-based flow cell in the detection of E. coli O157:H7 with 37-61% more impedance change for the frequency from 10Hz to 1MHz. The double IAM-based flow cell can be used to design a simple impedance biosensor for the sensitive detection of bacterial growth and their metabolites.     

多孔"类碳糊电极"的羧基化及其对Escherichia ocli O157:H7的检测

以吡咯为前驱体,羧基化碳纳米管、石墨粉为填料和碳酸钙微球为模板直接诱导合成,制备出一种高灵敏的多孔"类碳糊电极"生物电化学传感器,讨论了羧基化碳纳米管含量、银染时间对检测结果的影响.结果表明,最佳羧基化碳纳米管含量为8%,最佳银染时间为12 min,银的阳极溶出峰电流与E. ocli O157:H7浓度在1.0×104～1.0×106 cells/mL范围内呈线性关系,其线性回归方程为:IP=-5.582+1 972logC,相关系数(R2)为0.9912,检出限为5.1×103cells/mL,实现了对E. ocli O157:H7快速、准确地检测.     

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 fluorescein-tagged 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 micro g 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.     

Structure of the O-polysaccharide of Escherichia coli O60

Russian Chemical Bulletin - Structure of the O-polysaccharide (O-antigen) of Escherichia coli O60 was studied by sugar analysis, partial solvolysis with CF3CO2H, and 1D and 2D 1H and 13C NMR...     

Bead-Based Optical Immunoassay Using Quantum-Dot Labeling and Immunocomplex Dissociation for Detection of Escherichia coli O157:H7

An immunoassay for Escherichia coli O157:H7 using quantum-dot (QD) labeling and subsequent release of the QD labels from immunocomplexes has been developed. The assay principle is that anti-E. coli O157:H7 conjugated immunomagnetic beads are used to capture E. coli O157:H7; this is followed by the binding of QD labeled antibodies to form sandwich immunocomplexes (Bead-Cell-QD); a dissociation buffer then elutes QDs from immunocomplexes by denaturing antibody or lysing cell; the fluorescence signal of the eluted QDs is measured to quantify E. coli O157:H7. This proposed approach avoids the interference of bead autofluorescence in signal transduction and, thus, enhances the detection resolution, while keeping the fast magnetic separation and sandwich binding of two selective antibodies for high specificity.