Visual immunoprecipitate assay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.09): collaborative study

AOAC Official Method 996.09, Visual Immunoprecipitate Assay (VIP) for Escherichia coli O157:H7, was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by VIP assay and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment procedure and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 15 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level, where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 396 test portions and controls by both methods, for a total of 792 test portions. Of the 396 paired test portions, 75 were positive and 230 were negative by both the VIP and culture methods. Eleven test portions were presumptively positive by VIP and could not be confirmed culturally; 32 were negative by VIP, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the VIP method. There was no statistical difference between results obtained with the VIP for EHEC 8 h method and the culture method except for cooked beef, where the VIP had significantly higher recovery for one inoculation level.     

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.     

Reveal 8-Hour Test System for detection of Escherichia coli O157:H7 in raw ground beef, raw beef cubes, and iceberg lettuce rinse: collaborative study.

Five different food types were analyzed by the Reveal for E. coli O157:H7 8-Hour Test System (Reveal 8) and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method or the U.S. Department of Agriculture Food Safety Inspection Service (FSIS) culture method for the presence of E. coli O157:H7. A total of 27 laboratories representing academia and private industry in the United States and Canada participated. Food types were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. During this study, 1,110 samples and controls were analyzed by both the Reveal 8 and by BAM or FSIS by each of the collaborators (2,220 samples in total). For each set of samples, 740 were artificially inoculated with E. coli O157:H7, and 370 were uninoculated controls. The Reveal 8 detected 528 presumptive positives of which 487 were confirmed positive by the BAM culture method. In comparison, BAM and FSIS detected 489 of the 740 artificially contaminated samples as positive. In an additional in-house study performed only on chilled and frozen raw ground beef, 240 artificially inoculated samples were analyzed by both the Reveal 8 and by FSIS. The Reveal 8 detected and confirmed 104 samples as positive compared to 79 confirmed positive by FSIS.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC methods 996.09, Vip for EHEC and 996.10, assurance Eia EHEC with the reference culture method for the detection of Escherichia coli O157:H7 in beef

The Visual Immunoprecipitate Assay (VIP) method for the detection of enterohemorrhagic Escherichia coli O157:H7 (VIP for EHEC) and Assurance Enzyme Immunoassay (EIA) method for the detection of EHEC (EHEC EIA) are AOAC INTERNATIONAL Official Methods 996.09 and 996.10, respectively. A minor modification to the enrichment medium used in both methods has been developed. This modification, the BioControl modified EHEC medium (BioControl mEHEC) provides a more cost-effective procedure with performance equivalent to that of the cultural method for detection of E. coli O157:H7 in beef.     

Method extension study to validate applicability of AOAC Official Method 996.14 Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Assurance Listeria Polyclonal Enzyme Immunoassay (EIA) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 23 collaborators, representing government agencies, as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 550 test portions and controls was analyzed and confirmed, of which 207 were positive and 336 were negative by both methods. Six test portions were positive by culture, but negative by the EIA. Three test portions were negative by culture, but positive by the EIA. Two test portions were negative by EIA and by culture, but confirmed positive when EIA enrichment broths were subcultured to selective agars. The data reported here indicate that the Assurance Listeria EIA method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

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.     

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).     

Comparison of the Reveal 20-hour method and the BAM culture method for the detection of Escherichia coli O157:H7 in selected foods and environmental swabs: collaborative study.

Four different food types along with environmental swabs were analyzed by the Reveal for E. coli O157:H7 test (Reveal) and the Bacteriological Analytical Manual (BAM) culture method for the presence of Escherichia coli O157:H7. Twenty-seven laboratories representing academia and private industry in the United States and Canada participated. Sample types were inoculated with E. coli O157:H7 at 2 different levels. Of the 1,095 samples and controls analyzed and confirmed, 459 were positive and 557 were negative by both methods. No statistical differences (p <0.05) were observed between the Reveal and BAM methods.     

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06)

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.     

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.     

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.     

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.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.     

Escherichia coli O157:H7 detection limit of millimeter-sized PZT cantilever sensors is 700 cells/mL.

A composite self-excited millimeter-sized lead zirconate titanate (PZT) glass cantilever (2 mm x 1.8 mm; sensing area of 6 mm2) was fabricated for the detection of Escherichia coli (E. coli) O157:H7. The fundamental and second mode resonance in air was 10.95 +/- 0.05 kHz and 43.45 +/- 0.05 kHz, respectively. Affinity purified monoclonal antibody (anti-E. coli O157:H7) specific to the pathogen E. coli O157:H7 was immobilized at the cantilever glass tip, and then immersed in liquid containing the pathogen (70 to 7 x 10(7) cells/mL). The resonant frequency showed a reduction and reached a steady state shift of 0 +/- 5, 46 +/- 5, 260 +/- 5, and 1010 +/- 5 Hz corresponding to 0, 700, 7000, and 7 x 10(7) cells/mL. From the experiments conducted, the detection limit of the sensor was 700 cells/mL.     

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.     

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.     

Evaluation of the assurance GDS for E. coli O157:H7 method and assurance GDS for shigatoxin genes method in selected foods: collaborative study

A multilaboratory collaborative study was conducted to compare the Assurance GDS for E. coli 0157:H7 method and the reference culture methods for the detection of E. coli 0157:H7 in orange juice, raw ground beef, and fresh lettuce. A separate companion assay, the Assurance GDS for Shigatoxin Genes method was also evaluated with the same test portions. Fifteen laboratories participated in the study. A Chi square analysis of each of the 3 food types at the high, low, and uninoculated control levels was performed. For all foods, the Assurance GDS for E. coli O157:H7 method and the Assurance GDS for Shigatoxin Genes method were equivalent to or better than the reference methods.     

Simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes using oscillatory-flow multiplex PCR

An oscillatory-flow multiplex PCR method in a capillary microfluidic channel has been developed for the simultaneous determination of pre-purified DNA of multiple foodborne bacterial pathogens. The PCR solution passes three temperature zones in an oscillatory manner. The thermal stability and sample evaporation of the microfluidic device were investigated. Under controlled conditions, a highly efficient multiplex PCR was accomplished as demonstrated for the simultaneous amplifications of 278 bp, 168 bp, and 106 bp DNA fragments within 35 min after 35 cycles for simultaneous detection of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. This is much shorter than that of a conventional PCR machine. The detection limits of bacterial genome DNA for the three species are about 399, 314, and 626 copies per μL, respectively. This is comparable to those obtained with the conventional multiplex PCR. Consequently, the oscillatory-flow multiplex PCR technology holds good potential for rapid amplification and detection of nucleic acids of microbial foodborne pathogens.

Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.