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.     

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.     

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.     

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.     

Association of Escherichia coli O157:H7 with houseflies on a cattle farm

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 x 10(1) to 1.5 x 10(5) CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.     

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.     

Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples

An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min.     

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.     

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.     

Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 x 10(3) CFU g(-1) or <1 x 10(3) CFU ml(-1) and animals with E. coli O157 levels of > or =1 x 10(3) CFU g(-1) or > or =1 x 10(3) CFU ml(-1) in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (> or =1 x 10(3) CFU g of feces(-1)) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

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 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.     

Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on inactivation of Escherichia coli O157:H7 in raw-milk cheese

The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of Escherichia coli O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with E. coli O157:H7 at approximately 10(5) CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 10(6) CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10 degrees C in both cases. After 60 days, E. coli O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of E. coli O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated E. coli O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced E. coli O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated E. coli O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.     

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.     

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 electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.