Use of activated carbon coated with bentonite for increasing the sensitivity of pcr detection of Escherichia coli O157:H7 in Canadian oyster (Crassostrea gigas) tissue

A novel method for directly increasing the recovery of Escherichia coli O157:H7 and efficiently eliminating PCR inhibitors in oyster tissue without preenrichment was developed with the use of activated carbon coated with bentonite. The recovery of E. coli O157:H7 was significantly affected by the amount of bentonite used to coat the activated charcoal and the pH value of sample preparations. When 4.2 g of activated carbon were coated with 0.4 g of bentonite and seeded oyster samples were adjusted to a pH of 5.0, a high recovery of E. coli O157:H7 (91.6+/-4.4%) was obtained. Activated carbon, coated with bentonite, allowed the PCR detection of 1.5 x 10(2) CFU/g of oyster tissue which was equivalent to 30 genomic targets per PCR reaction. Without the use of activated carbon coated with bentonite, the minimum level of detection was 1.5 x 10(5) CFU/g of oyster tissue, which is equivalent to 3.0 x 10(4) genomic targets per PCR reaction. Three commercial DNA purification systems were used for comparison. The limit of detection with the Wizard DNA Clean-Up System and the Chelex(R)100 Resin was 1.5 x 10(3) CFU/g of oyster tissue which was equivalent to 3.0 x 10(2) CFU/PCR reaction. The QIAamp DNA Mini Kit resulted in a detection limit of 5 x 10(2) CFU/g of oyster tissue which was equivalent to 5 x 10(2) genomic targets per PCR reaction. The use of activated carbon coated with bentonite is an inexpensive method for removal of PCR inhibitors from tissue samples prior to the release of DNA from target cells resulting in relatively low numbers of target cells detected without enrichment.     

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.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

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.     

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.     

Using oligonucleotide-functionalized Au nanoparticles to rapidly detect foodborne pathogens on a piezoelectric biosensor

A circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method, was used for real-time detection of a foodborne pathogen, Escherichia coli O157:H7. A synthesized thiolated probe (Probe 1; 30-mer) specific to E. coli O157:H7 eaeA gene was immobilized onto the piezoelectric biosensor surface. Hybridization was induced by exposing the immobilized probe to the E. coli O157:H7 eaeA gene fragment (104-bp) amplified by PCR, resulting in a mass change and a consequent frequency shift of the piezoelectric biosensor. A second thiolated probe (Probe 2), complementary to the target sequence, was conjugated to the Au nanoparticles and used as a "mass enhancer" and "sequence verifier" to amplify the frequency change of the piezoelectric biosensor. The PCR products amplified from concentrations of 1.2 x 10(2) CFU/ml of E. coli O157:H7 were detectable by the piezoelectric biosensor. A linear correlation was found when the E. coli O157:H7 detected from 10(2) to 10(6) CFU/ml. The piezoelectric biosensor was able to detect targets from real food samples.     

Comparison of primers for the detection of pathogenic Escherichia coli using real-time PCR

AIMS: To evaluate PCR primers for the detection of pathogenic Escherichia coli in a real-time PCR assay and determine their utility in produce irrigation water testing. METHODS AND RESULTS: Three previously published PCR primer sets and one set designed for this study were tested for their ability to produce amplification products for several pathogenic E. coli serotypes from whole cells as template. Two of the previously published primer sets were chosen for real-time PCR detection limit determination. The coneaeA and PEH detection limit of E. coli O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect E. coli O157:H7 in sprout irrigation water, the water required dilution due to PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between 10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively. CONCLUSIONS: The primer set coneaeA was able to produce an amplification product from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR detection of pathogenic E. coli in diluted sprout irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: The necessity of a dissociation analysis to distinguish positive samples from those with fluorescence of random dsDNA generation for real-time PCR in a complex background was established.     

EVALUATION OF 5'NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Two 5'nuclease-based PCR methods (PCR-LS-50B and PCR-7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR-7200 method was able to detect E. coli O157:H7 at ± 10² CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR-7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR-LS-50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR-based detection techniques for E. coli O157:H7, however, the 5'nuclease-based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.     

Evaluation of a real-time PCR kit for detecting Escherichia coli O157 in bovine fecal samples

A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g(-1) in artificially inoculated bovine feces following enrichment.     

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.     

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.     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

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.     

Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction

A new lysis solution designated TZ, consisting of 2.0% Triton X-100 plus 2.5 mg sodium azide/ml in 0.1 M Tris-HCl buffer at pH 8.0, yielded higher levels of genomic DNA from Escherichia coli O157:H7 cells compared with a number of other commonly used cell lysis methods. Ethidium bromide stained DNA bands resulting from PCR amplification of target DNA from 100 CFU of E. coli O157:H7 were readily detected following electrophoresis of agarose gels. In contrast, conventional cell lysis methods failed to detect target DNA from 100 CFU after PCR amplification. The new solution was highly effective for lysing cell suspensions of Salmonella enteritidis, Pseudomonas putida, Lysteria monocytogenes and Psychrobacter immobilis.     

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.     

Potential pitfalls in the quantitative molecular detection of Escherichia coli O157:H7 in environmental matrices

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene-based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

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.     

QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.     

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10ml of brain heart infusion (BHI) broth for 18h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.