Strategies for the detection of Escherichia coli O157:H7 in foods

Summary, 419 Introduction, 419 Epidemiology and occurrences, 420 Methods of detection, 420 Conventional methods, 420 Immunomagnetic separation, 421 Immunological detection, 421 Enzyme-linked immunosorbent assay, 421 Nonenzymatic immunoassays, 421 PCR-based detection methods, 422 The BAX® automated PCR system, 423 Biosensors, 423 Fluorescence and microscopy, 424 Emerging technologies, 424 Microarrays, 424 Molecular beacons, 424 Integrated systems, 425 Concluding remarks, 425 References, 427 This review assesses the various methods used to detect Escherichia coli O157:H7 in foods. As this organism has been involved in many outbreaks of disease, it is essential to develop a rapid, yet reliable, method of detection. Conventional methods such as culturing and biochemical tests are covered, followed by a discussion of immunological methods. Both enzymatic and nonenzymatic approaches are discussed, and commercially available kits based on these principles are described. PCR has allowed the rapid amplification of very small numbers of organisms and standard PCR along with multiplex and real-time PCR are discussed. Biosensors and microarrays can provide real-time detection and the current status of each of these is reviewed. It is believed that molecular beacons and integrated systems (lab-on-a-chip) can offer potential advantages for the detection of this pathogen and both are analysed. Drawbacks and advantages of each method described are considered throughout the article. Escherichia coli was first isolated in 1885 from children's faeces by the German bacteriologist Theodor Escherich. It is a normal commensal organism of the gastrointestinal tract of human beings and, although, generally harmless, it can cause a number of infections such as Gram-negative sepsis, urinary tract infection, pneumonia in immunocompromised patients and meningitis (Adams and Moss 1995). Strains of E. coli were first recognized as a cause of enteritis by workers in England investigating summer diarrhoea in infants in the early 1940s (Adams and Moss 1995). Until 1982, three major strains were described: enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC). In 1982, E. coli serotype O157:H7 was implicated in two outbreaks of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). This organism has been classified as verocytotoxigenic E. coli (VTEC). Haemorrhagic colitis is characterized by abdominal pain, watery diarrhoea followed by bloody diarrhoea. HUS occurs in all age groups but is more common in infants and young children. It is the most important complication of infection by E. coli O157:H7 and is characterized by the sudden onset of haemolytic anaemia with fragmentation of red blood cells, thrombocytopenia and acute renal failure after acute gastroenteritis. The gastrointestinal disease may be severe, with HC being presented, and the central nervous system, pancreas, lungs and heart also being affected (Fitzpatrick 1999). Food is one of the most important sources of VTEC infection and some of those implicated are hamburger meat, bruised apples, unpasteurized apple juice and milk, potatoes, lettuce, unchlorinated water and mayonnaise. Cattle are considered to be one of the major sources of O157:H7, which is spread through faecal contamination of food. The bovine population is also an important reservoir as shedding occurs intermittently, the timing of which is difficult to predict. Thus, human beings may be infected at any time and all measures should be taken to reduce the risk to public health. The first major occurrences were in the USA in 1982 which involved hamburgers from fast food chains in Oregon and Michigan (Snyder 1998). In 1988, 30 students at a high school in Minnesota fell ill after consuming partially-cooked beef patties, and in late 1992 and early 1993, four deaths in four states (Washington, Idaho, California and Nevada) were recorded. These were once again attributed to hamburgers. In 1996, unpasteurized apple juice was implicated in an outbreak in California. Also in that year, an outbreak in Lanarkshire, Scotland claimed 20 lives at a nursing home. This was as a result of the victims consuming beef contaminated with the organism (Bell and Kyriakides 1998). In August 1997, Hudson Foods recalled 25 million pounds of ground beef after an E. coli outbreak was traced to its plant in Nebraska (Snyder 1998). In 1999, a serious outbreak affected New York state where 1000 people were affected with two deaths recorded (Charatan 1999). In this incident, the source was found to be a contaminated well at the Washington County Fair in upstate New York. Runoff from cow manure after torrential rain contaminated the well and water consumption through products such as ice, snow cones and lemonade was the likely exposure route (Charatan 1999). In 2000, the major outbreak was in Walkerton, Ontario where seven people died and 2300 became ill as a result of infected water supplies. In 2001, 100 cases of poisoning because of E. coli was reported at nursing homes in Ontario while in 2002 there were further concerns about contaminated water in southern Ontario. In general, the infected patients are usually vulnerable members of the community such as the very young or very old and immunocompromised individuals. The infective dose of E. coli O157:H7 is 50–100 organisms and the incubation period to the onset of diarrhoea can vary from 1 to 8 days (Singleton 1995). The satisfactory microbiological quality for E. coli O157 is <20 CFU g−1 with the acceptable range being 20 to <100 CFU g−1 (Gilbert et al. 2000). However, it is the opinion of the Advisory Committee for Food and Dairy Products (ACFDP) of the UK that ready-to-eat foods should be free from E. coli O157 and other VTEC organisms (Gilbert et al. 2000). An important perspective is the Public Health aspect associated with outbreaks of disease caused by E. coli O157:H7. The Centers for Disease Control in the USA has estimated that 76 million people suffer from food-borne illnesses annually with 325 000 being admitted to hospitals of whom more than 5000 die. It has been estimated that the yearly cost of these illnesses is US $5–6 billion in direct medical expenses and lost productivity. Escherichia coli O157:H7 causes 73 000 illnesses and 61 deaths per year in the USA (CDC 2003). In the UK, the Health Protection Agency (formerly, the Public Health Laboratory Service) has indicated that in 2001, there were 85 468 food poisoning notifications, which represent a sixfold increase from 1982. Of these, 768 were because of E. coli O157:H7 (Health Protection Agency 2003). Thus, it is clear that rapid and sensitive methods for this organism are required. Conventional methods have included plating and culturing, and the use of biochemical tests. With respect to E. coli O157:H7, detection has been carried out by the use of sorbitol MacConkey agar (SMAC), which consists of bile salts, a carbohydrate source, sorbitol and an indicator. Under normal laboratory conditions, O157:H7 does not ferment sorbitol. If O157:H7 is present, colourless colonies will appear while other Enterobacteriaceae will show up as pink colonies. Early work on the use of SMAC found that, for E. coli O157:H7, there was a high level of accuracy and sensitivity (March and Ratnam 1986). Enrichment broths containing peptone, vancomycin, cefixime, cefsulodin and potassium tellurite are also used to enhance the detection of the organism by providing nutrients which allow the specific organisms to produce more colonies. In recent years, modified agar methods have been described for different applications. Silk and Donnelly (1997) have reported that by using trypticase soya agar, acid-injured E. coli O157:H7 in autoclaved apple cider were detected at higher sensitivities than other media. The apple cider had a pH of 3·2 which will injure the bacteria and the method was developed to identify viable organisms after 72 h in the cider. The authors have indicated that special recovery steps are needed when analysing acidic foods, which may contain the bacterium. A universal pre-enrichment (UP) medium has been developed for the simultaneous recovery of E. coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella enterica serotype typhimurium (Thippareddi et al. 1995). It was found that the addition of oxyrase enhanced the growth of the organisms being investigated. Even injured bacteria were recovered from inoculated food samples such as turkey ham, mayonnaise and ground beef. This approach may prove useful where few organisms are implicated in causing disease. An interesting development has been the combined use of commercially available rainbow agar O157 and PCR. This was used to detect the organism in raw meat with the rainbow agar being selective and sensitive for the screening of E. coli O157:H7 from artificially and naturally contaminated meat samples in 24 h (Radu et al. 2000). Isolates suspected of containing the pathogen were amplified by PCR with this aspect adding a further 6–8 h to the analysis time. This method may be considered as time-consuming. Several biochemical and other tests (the IMViC tests) can be used to differentiate E. coli from other Enterobacteriaceae. These include the ability to produce indole from tryptophan (I), sufficient acid to reduce the medium pH below 4·4, the break point of the indicator methyl red (M), acetoin (V) and the ability to utilize citrate (C) (Adams and Moss 1995). Usually, the first preliminary test involves the use of the API system (bioMerieux, Paris, France), which can identify enterobacteria in 4 h based on the use of biochemical tests. Furthermore, the system can be used to detect other organisms such as Staphylococcus, Candida, Streptococcus and Campylobacter. Latex agglutination reactions are simple and easy to use, and are used to detect the presence of either antibody or antigen in a sample. These are attached to latex beads and if the corresponding antigen or antibody is present, the latex beads will agglutinate when mixed with the sample being investigated. The separation and detection of a specific microbial species from a mixed culture, such as food, by plating on selective media is usually inefficient without pre-enrichment steps (Safarik et al. 1995). One approach towards solving this problem is to use a specific magnetic separation of the target organism directly from the sample or pre-enrichment medium. Most of the particles used for these separations are super-paramagnetic, i.e. they only exhibit magnetic properties in the presence of an external magnetic field. They can be easily removed by a magnetic separator (Safarik et al. 1995). The most common magnetic carriers are Dynabeads® (Dynal, Oslo, Norway) which are polystyrene-based particles ranging from 2·8 to 4·5 μm. It is also possible to obtain coated Dynabeads® with covalently immobilized streptavidin or secondary antibodies against selective primary antibodies. In the direct IMS technique, immunomagnetic particles specific for the target organism are suspended in the mixed cell suspension. After incubation, the particles with bound target cells are separated from the suspension with a magnetic particle separator, the remaining suspension is removed and magnetic particles are washed several times (Safarik et al. 1995). The indirect approach involves the addition of primary antibodies to the bacterial suspension and binding of the target cell-surface structures. Magnetic particles with immobilized secondary antibodies are then added. After the interaction of the primary and secondary antibodies, the entire complex is removed from the suspension by a magnetic separator (Safarik et al. 1995). Immuno-detection has become a widely used approach for E. coli O157:H7 because it allows for sensitive and specific detection. In this section, we will describe some of the methods, which have been developed. Enzyme-linked approaches are very popular and some interesting methods have been reported in the literature. One of these involves the use of enzyme-linked immunomagnetic electrochemistry. This involves the sandwiching of bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody (Gehring et al. 1999). The beads were attached to the surface of magnetized graphite ink electrodes in a multiwell plate format. The substrate was then added and the electroactive product generated was measured by square-wave voltammetry. Detection of 4·7 × 103 cells ml−1 was possible in ca 80 min. A chemiluminescence enzyme immunoassay has been developed which uses different E. coli O157 serotypes (Kovacs and Rasky 2001). Tenfold dilutions of 24 h broth cultures of the test strains were performed and the detection limit was found to be 103–104 cells ml−1. Fratamico and Strobaugh (1998) compared enzyme-linked immunosorbent assay (ELISA) with the direct immunofluorescent filter technique (DIFT) and multiplex PCR for the detection of the bacterium in beef carcass wash water. They found that, following a 4 h enrichment culturing, ELISA could detect 100 colony forming units (CFU) ml−1 while DIFT detected 0·1 CFU ml−1 and multiplex PCR was 1 CFU ml−1. This gives excellent sensitivities but a major drawback is the lengthy enrichment procedure. Several other types of immunoassays have been developed for the detection of E. coli O157:H7 with a few interesting ones being chosen for study. Mansel Griffiths’ group has used immunomagnetic separation (IMS) and a fluorescently stained bacteriophage to detect the pathogen in broth (Goodridge et al. 1999a). In combination with flow cytometry, the fluorescent-bacteriophage assay (FBA) was able to detect ca 100 cells ml−1. These investigators have also used this approach to detect E. coli O157:H7 in inoculated ground beef and raw milk (Goodridge et al. 1999b). It was reported that 2·2 CFU g−1 of artificially contaminated ground beef were detected after a 6-h enrichment. In milk, the detection limit was 10–100 CFU ml−1, following a 10-h enrichment procedure. The FBA may be useful as a method for the preliminary detection of the organism in food but further research into the specificity and applications to other foods are required before it can be widely adopted. A solid phase fluorescent capillary immunoassay has also been developed (Czajka and Batt 1996). A soft-glass capillary tube served as a solid support for adsorption of heat-killed bacteria. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used with the bound antigen–antibody complex being detected by avidin labelled with a fluorescent cyanine dye. For ground beef, the detection limit was 1 CFU 10 g−1 after enrichment for 7 h while for apple cider it was 0·5 CFU ml−1 after a 7-h enrichment. In a similar approach, a time-resolved fluorescence immunoassay (TRFIA) using IMS was used to detect organisms in apple cider (Yu et al. 2002). The TRFIA used a polyclonal antibody bound to immunomagnetic beads as the capture antibody, with the same antibody labelled with europium as the detection antibody. The authors indicate that the limit of detection of the assay was 103 cells with 10–100 CFU ml−1 detected in 6 h. This appears comparable with the results obtained by the solid phase fluorescent immunoassay. In recent years, PCR has become very important as a technique for the detection of bacteria. The main reason for this is that the DNA from a single bacterial cell can be amplified in about 1 h, which is very rapid compared with the methods described previously. However, the method can also amplify dead cells and care must be taken in designing the experiments. Furthermore, this makes data interpretation complex and it is an issue that has to be addressed as it has long-range implications from a legal perspective. This is especially true in the EU where new legislation will be introduced shortly. Escherichia coli may be found in a number of unrelated environments and its ability to survive beyond a host, which will permit re-infection, is an important feature of its life cycle (Jaykus 2003; Winfield and Groisman 2003). This is of paramount importance and must be considered when designing and developing real-time detection methods. In this account, we will describe a few of the approaches involving this technique to detect E. coli O157:H7. A PCR assay targeting the 3′-end of the eae gene of E. coli O157:H7 was found to be specific, with sensitivity being 1 pg DNA or 103 CFU PCR per reaction (Uyttendaele et al. 1999). Furthermore, studies were carried out to determine the effect of the food matrix and sample preparation method on PCR detection of nonviable cells using heat-killed bacteria in ground beef. Sample preparation methods included centrifugation, buoyant density centrifugation (BDC), IMS, chelex extraction and swabbing. It was found that IMS was the only method which did not produce false-positive results, provided the number of cells were below 108 CFU g−1. Above this number, this method also produced false positives which is a severe limitation of this approach. Several variations of the standard PCR have recently appeared and these have assisted in producing more sensitive detection methods. Of these, multiplex PCR and real-time PCR are proving to be the most popular. The former allows several targets to be co-amplified in one PCR by combining or ‘multiplexing’ primer pairs (Newton and Graham 1997). Real-time PCR allows reactions to be characterized by the time when amplification of the PCR product is first detected by use of a fluorogenic probe (Livak 2000). Hu et al. (1999) have described a multiplex PCR using five sets of primers which amplified regions of eae A, slt1, slt2, fli C and rfb E genes. By analysing 82 E. coli strains (both O157:H7 and non-O157:H7), it was found that the assay could successfully distinguish serotype O157:H7 from other serotypes. In this instance, bovine faeces was also studied. A multiplex PCR was used to detect the pathogen in soil and water (Campbell et al. 2001). Soil and water samples were spiked with E. coli O157:H7 and enriched prior to PCR. The researchers reported detection limits of 1 CFU ml−1 drinking water and 2 CFU g−1 soil. An interesting aspect was that starvation of the bacteria for 35 days before addition to soil did not affect the detection of initial cell numbers as low as 10 CFU g−1 soil. Results were obtained in one working day. Finally, a reverse transcriptase PCR (RT-PCR) has been developed to detect viable E. coli O157:H7. RT is an enzyme which is capable of synthesizing single-stranded DNA from RNA in the 5′–3′ direction (Newton and Graham 1997). Yaron and Matthews (2002) studied several genes (rfb E, fli C, slt1, slt2, mob A, eae A, hly and 16S rRNA) as indicators for viability. They found that rfb E, slt1, hly and 16S rRNA amplicons were detected in all growth phases with the products of 16S rRNA, mob A, rfb E and slt1 being readily visualized in RNA isolated from viable cells. However, the 16S rRNA target was not amplified after heat treatment. The authors suggest that the rfb E gene is the most suitable target for detecting viable bacteria. Without pre-enrichment, RT-PCR can detect 107 CFUs of the organism. This represents an advance over those PCR approaches, which require enrichment as it reduces the time required for analysis while retaining a high sensitivity. The BAX® system with automated detection was developed by Du Pont Qualicon (Wilmington, DE, USA) and it allows for the rapid detection of bacteria in raw ingredients, finished products and environmental samples (Qualicon 2001). It uses DNA amplification followed by gel electrophoresis to determine the presence or absence of a specific target (Fritschel 2001). Primers, DNA polymerase and deoxynucleotides for PCR, a positive control and an intercalating dye are combined into one tablet. Additionally, an instrument, which integrates the amplification and detection steps, has been developed. This uses an array of 96 blue light-emitting diodes as the excitation source and a photomultiplier tube to detect the fluorescent signal, which is produced (Fritschel 2001). The assay tablets are hydrated with lysates from foods after overnight enrichment. The instrument does thermal cycling on the samples and then analyses the melting curve to determine if the target is present. Several papers have appeared in the last 4 years which evaluated the BAX® PCR system. Shearer et al. (2001) studied 15 food samples, including cabbage, radish, mushroom, tomato and mango, for E. coli O157:H7, and reported that BAX was more sensitive than culturing for some samples. However, they reported that both BAX and culture methods were unable to recover low numbers of the bacteria from alfalfa sprouts. In another study, it was claimed that the BAX system was better than conventional methods with the former having a detection rate of 96·5% compared with 39% for the best culture method (Johnson et al. 1998). The BAX is ideal for the identification of E. coli O 157:H7 in various foods but it does not provide quantification of the organisms present. One of the major problems in developing a rapid test for E. coli O157:H7 is that a number of steps are usually involved, including lengthy enrichment procedures. Thus, biosensors are becoming more commonplace but there are still several issues remaining to be solved. These will be discussed in this section and in the Conclusion along with the attempts, which have been made to develop biosensors for this pathogen. An evanescent-wave fibre optic biosensor was used to detect the bacterium in 10 and 25 g ground beef samples (Demarco and Lim 2002). The biosensor uses a 635-nm laser diode to direct light onto optical fibre probes, which generates the evanescent wave. Fluorescent molecules within the evanescent field are excited and a part of the emission re-couples into the fibre probe. A photodiode detects and quantifies the fluorescent signal. A sandwich immunoassay was utilized which allowed the detection of 9·0 × 103 CFU g−1 for 25 g samples and 5·2 ×102 CFU g−1 for the 10-g sample. The authors reported that no false positives were obtained with results being obtained 25 min after sample processing. Krull's group has used a fibre optic biosensor operated in a total internal reflection format to assist in the detection of genomic DNA from coliforms, including E. coli (Almadidy et al. 2002). Genomic DNA was extracted and sonicated to prepare 300-mer fragments. It was reported that detection of fragments containing the lac Z sequence was obtained in ca 20 s by fluorescence measurements. Deisingh and Thompson (2001) have described a PCR-acoustic wave sensor combination to detect sequences of E. coli O157:H7. PCR was used to amplify a 509 base sequence unique to the pathogen and, in real time, a biotinylated probe was attached to the surface of the sensor. This was achieved by using the biotin–neutravidin interaction, the latter having been pre-adsorbed to the surface of the sensor. The authors have suggested that this approach may be suitable for detecting the organism in food, water and clinical samples. It has also been proposed that this method may be incorporated into an integrated system for rapid PCR-based DNA analysis but this will require much more research before this aim may be achieved. Several papers reporting on the use of surface plasmon resonance (SPR) have begun to appear in the literature. A BIACore biosensor (Fig. 1) using antibodies against E. coli O157:H7 was found to have a detection limit of 5 × 107 CFU ml−1 (Fratamico et al. 1997). However, this, does not compare well with other biosensor methods as described above. Karube's group has used chimeric oligonucleotides consisting of 21 bases of RNA with six bases of DNA at the 3′-hydroxyl terminus and a recombinant thermostable DNA polymerase to amplify DNA fragments by PCR (Miyachi et al. 2000). A chimeric RNA–DNA primer can serve as a primer in conventional PCR. The PCR products were then examined by SPR, which allowed the differentiation of the O157:H7 strains from other bacteria. Medina (2002) has reported on the use of SPR to investigate the binding reactions of immobilized E. coli O157:H7 with extracellular components such as collagen, fibronectin, laminin and glucoaminoglycans. A model system was used to evaluate the inhibition of collagen–laminin binding on the sensor surface with polysulphated polysaccharides (heparan sulphate and carrageenans). She indicated that carrageenans inhibited 71–99% of binding while heparan sulphate inhibited 39–41%. These studies were performed to allow a rapid assessment of compounds for carcass treatment to inhibit or detach pathogens from meat. Schematic of the BIACORE surface plasmon resonance spectrometer (reprinted from: G. Ramsay, Commercial Biosensors, 1997, by permission of John Wiley and Sons Inc.) Laser-induced fluorescence coupled with flow cytometry was used to detect E. coli O157:H7 in ground beef (Johnson et al. 2001). The authors reported that this approach offers several advantages over currently available techniques including: (i) it is able to examine large quantities of food or water in real time, (ii) it can detect single organisms whereas other methods may require more than 104 microbes, (iii) the method is automated and (iv) it is specific for the organisms being studied. It is believed that this system can provide the sensitivity and specificity required for the detection of pathogenic bacteria. Recently, a study investigating the effect of treating E. coli O157:H7 cells labelled with an enhanced green fluorescent protein (EGFP) plasmid was carried out (Burnett and Beuchat 2002). The cells were treated with chlorine, hydrogen peroxide and acetic acid, and changes in fluorescent intensity were observed. Confocal scanning laser microscopy was also performed. The researchers indicate that EGFP used to label the pathogen may not be suitable for microscopic differentiation of viable and dead cells after treatment with sanitizers. It was found that SYTOX Green (Molecular Probes, Eugene, OR, USA) was preferable for the detection of dead cells while antibodies labelled with Alexa Fluor 594 (Molecular Probes) is the substance of choice for the determination of total (dead and viable) cell counts. In this section, we will consider three approaches which are having great impact towards producing rapid and sensitive detection methods for E. coli O157:H7. These are the development of integrated systems (lab-on-a-chip), the use of molecular beacons and the production of microarrays. However, it should be noted, that none of these methods is free from problems and the organism continues to provide a major challenge in detection. Microarrays allow thousands of specific DNA or RNA sequences to be detected simultaneously on a small glass or silica slide about 1–2 cm2 (Aitman 2001). This has allowed more rapid analyses to take place but there are drawbacks to the use of this technology. Microarray instruments are expensive, of limited availability and require specialist knowledge and training to extract useful information from the huge amount of data generated. Notwithstanding these problems, papers are starting to appear which use this technology to detect E. coli O157:H7. Call and co-workers (2001) have reported on the use of multiplexed PCR and nucleic acid microarrays to attempt specific and sensitive detection. The array was composed of 25–30-mer oligonucleotide probes complementary to four targets (intimin, slt-1, slt-2 and haemolysin A). Target DNA was amplified by multiplex PCR, the amplicons being hybridized to the array without any purification. It has been claimed that the array is 32 times more sensitive than gel electrophoresis and was capable of detecting 1 fg of genomic DNA. The authors have further reported that by using a combination of immunomagnetic capture, PCR and a microarray, 55 CFU ml−1 of bacteria in chicken carcass wash water were detected without the need for pre-enrichment. In a later paper, this group has described the development of an electromagnetic flow cell and fluidics system for the automated IMS of E. coli O157:H7 from poultry carcass rinse (Chandler et al. 2001). No pre-enrichment was necessary and high porous nickel foam was used to enhance the magnetic field gradient within the flow path to ensure immobilization of the immunomagnetic particles throughout the fluid. Bacterial cells were isolated directly from poultry carcass rinse with a 39% recovery efficiency at 103 CFU ml−1. Molecular beacons (MBs) fluoresce upon hybridization to their complementary DNA target. The essential feature of these probes is their stem-loop structure (Brown et al. 2000). The stem is constructed of two short oligodeoxynucleotide arms, one terminally labelled with a fluorophore and the other with a quencher (Fig. 2). Hybridization of the loop to the target causes the stem to open with the result that the fluorophore and quencher are no longer close to each other. This leads to the emission of fluorescence (Brown et al. 2000). A modification of this approach involves supporting the molecular beacons on solid glass particles. These beacons exhibit similar properties to the soluble versions (Brown et al. 2000). Principle of detection of hybrids with molecular beacons (reprinted from: M.L.M. Anderson, Nucleic Acid Hybridization, Bios Scientific, 1999. By permission of the publisher) The use of MBs in the detection of E. coli O157:H7 is now beginning to have success and a few papers have been published recently. McKillip and Drake (2000) used a beacon to detect the pathogen in skimmed milk during PCR amplification of DNA. The probe was designed to hybridize to a region of the slt2 gene and to fluoresce when the stem-loop structure became ‘open’ upon hybridization. The u