Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10(2) CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.     

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.     

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

The purpose of this study was to apply our rapid, integrated double enrichment 5' nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.     

Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains

AIMS: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format. METHODS AND RESULTS: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E. coli O157. A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E. coli (EHEC) O157 strains and also with two enterotoxigenic E. coli O157 strains. Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected. CONCLUSION: A MAb-based sELISA to detect E. coli O157 was produced. Its application to field samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-specific interference of the cross-reacting strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay produced is not wholly specific to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli O157:H7 in apple cider.

An immunoassay based on immunomagnetic separation and time-resolved fluorometry was developed for the detection of E. coli O157:H7 in apple cider. The time-resolved fluorescent immunoassay (TRFIA) uses a polyclonal antibody bound to immunomagnetic beads as the capture antibody and the same antibody labeled with europium as the detection antibody. Cell suspensions of 10(1) to 10(8) E. coli O157:H7 and K-12 organisms per ml were used to test the sensitivity and specificity of the assay. The sensitivity of the assay was 10(3) E. coli O157:H7 cells with no cross-reaction with K-12. Pure cultures of E. coli O157:H7 (10(1) to 10(5) CFU/ml) in apple cider could be detected within 6 h, including 4 h for incubation in modified EC broth with novobiocin and 2 h for the immunoassay. When apple cider was spiked with 1 to 10(3) CFU/ml of E. coli O157:H7 and 10(6) CFU/ml of K-12, our data show that the high level of K-12 in apple cider did not impede the detection of low levels of O157:H7. The minimum detectable numbers of cells present in the initial inoculum were 10(2) and 10(1) CFU/ml after 4- and 6-h enrichment. The TRFIA provides a rapid and sensitive means of detecting E. coli O157:H7 in apple cider.     

Comparison of an immunochromatographic method and the TaqMan E. coli O157:H7 assay for detection of Escherichia coli O157:H7 in alfalfa sprout spent irrigation water and in sprouts after blanching

An immunochromatographic-based assay (Quixtrade mark E. coli O157 Sprout Assay) and a polymerase chain reaction (PCR)-based assay (TaqMan E. coli O157:H7 Kit) were used to detect Escherichia coli O157:H7 strain 380-94 in spent irrigation water from alfalfa sprouts grown from artificially contaminated seeds. Ten, 25, 60, or 100 seeds contaminated by immersion for 15 min in a suspension of E. coli O157:H7 at concentrations of 10(6) or 10(8) cfu/ml were mixed with 20 g of non-inoculated seeds in plastic trays for sprouting. The seeds were sprayed with tap water for 15 s every hour and spent irrigation water was collected at intervals and tested. E. coli O157:H7 was detected in non-enriched water by both the TaqMan PCR (30 of 30 samples) and the immunoassay (9 of 24 samples) in water collected 30 h from the start of the sprouting process. However, enrichment of the spent irrigation water in brain heart infusion (BHI) broth at 37 degrees C for 20 h permitted detection of E. coli O157:H7 in water collected 8 h from the start of sprouting using both methods, even in trays containing as few as 10 inoculated seeds. The TaqMan PCR assay was more sensitive (more positive samples were observed earlier in the sprouting process) than the immunoassay; however, the immunoassay was easier to perform and was more rapid. At 72 h after the start of the sprouting process, the sprouts were heated at 100 degrees C for 30 s to determine the effectiveness of blanching for inactivation of E. coli O157:H7. All of the 32 samples tested with the TaqMan assay and 16 of 32 samples tested with the Quixtrade mark assay gave positive results for E. coli O157:H7 after enrichment of the blanched sprouts at 37 degrees C for 24 h. In addition, the organism was detected on Rainbow Agar O157 in 9 of 32 samples after 24 h of enrichment of the blanched sprouts. In conclusion, E. coli O157:H7 was detected in spent irrigation water collected from sprouts grown from artificially contaminated seeds by both the TaqMan and Quixtrade mark assays. The data also revealed that blanching may not be effective to completely inactivate all the E. coli O157:H7 that may be present in sprouts.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.     

A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.

Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.     

Comparison of fluorogenic and chromogenic assay systems in the detection of Escherichia coli O157 by a novel polymyxin-based ELISA

AIMS: Different indicator enzymes and fluorogenic or chromogenic substrates were compared as detector systems in a novel polymyxin-based enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157 lipopolysaccharide (LPS) antigens. METHODS AND RESULTS: An ELISA system was developed using polymyxin immobilized in the wells of a microtitre plate as a high-affinity adsorbent for E. coli O157 LPS antigens, which were immunoenzymatically detected using anti-E. coli O157 antibody-enzyme conjugates. With peroxidase as the indicator enzyme the fluorogenic substrates Amplex Red and QuantaBlu produced only slight improvement in the performance characteristics of the polymyxin-ELISA compared with the use of the chromogenic substrate tetramethylbenzidine (TMB). On the other hand, with alkaline phosphatase as the indicator enzyme a pronounced improvement in assay performance was noted using the fluorogenic substrate Attophos compared with the chromogenic substrate p-nitrophenylphosphate. CONCLUSIONS: The detection system exhibiting the best characteristics with respect to cost, ease of use and overall performance in the detection of E. coli O157 in enrichment cultures from a variety of solid foods was based on the use of peroxidase as the indicator enzyme with the chromogenic substrate TMB. SIGNIFICANCE AND IMPACT OF THE STUDY: The polymyxin-ELISA provides a rapid, simple and inexpensive assay system for the detection of E. coli O157 in foods.     

Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 x 10(3) in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.