INTRODUCTION

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Verotoxigenic Escherichia coli (VTEC) was discovered by Konowalchuk and colleagues during the late 1970s in Canada (11), whereas enterohemorrhagic E. coli (EHEC) O157:H7 was first isolated in 1975 and was identified as an important food-borne bacterial pathogen in 1982 after two outbreaks of unusual gastroenteritis occurred in the states of Oregon and Michigan (20). The diseases caused by VTEC include bloody diarrhea, hemolytic-uremic syndrome, hemorrhagic colitis, and thrombotic thrombocytopenic purpura (18). The mechanism by which VTEC causes human illness is not fully understood; however, several factors are known to be associated with its virulence. One of these factors is the production of one or more verotoxins (VTs) (20).

According to their antigenic diversity, VTs are divided into two groups, VT1 and VT2. The former can be neutralized by the antiserum against Shiga toxin produced by Shigella dysenteriae type 1, whereas the latter cannot be neutralized by the same serum (19). The structural genes for VT1 and VT2 are bacteriophage encoded (26, 29) and share 55% overall nucleotide sequence homology (8).

VTEC infection can be transmitted by either person-to-person contact or the consumption of contaminated foods (20). Although foods such as mayonnaise (7, 23), apple cider (2, 16), yogurt (17), and cheese (1) have been implicated in VTEC infections, outbreaks of VTEC are commonly associated with undercooked ground beef and raw milk (20).

Studies have shown that cattle are a major reservoir for VTEC, and clinically healthy VTEC carriers may serve as the source of the contamination of processed meats by introducing the microorganism into processing plants (10). This explains why retail meats from diverse geographical locations tested positive for the presence of E. coli O157:H7 (3, 12, 24).

The most prevalent serotype of VTEC associated with human hemolytic-uremic syndrome is O157:H7. However, other serotypes have also been implicated in human VTEC infections (10).

Although there are reliable immunoassays for VTs, the toxin(s) must be expressed to allow detection. Therefore, genetic detection may be advantageous in that such methods would detect potentially pathogenic strains irrespective of assay conditions. Detection of VTEC by conventional PCR has been reported (14, 21, 30), but interference with the PCR has been observed when the technique was applied directly to foods (13, 22). Attempts have been made to remove the inhibitors to PCR present in food samples by ether extraction, column purification (27), or the addition of bovine serum albumin, proteinase inhibitors (22), and Tween 20 (27). However, no single method has been found to be ideal.

Magnetic capture-hybridization PCR (MCH-PCR) was initially used to overcome the inhibitory effect of humic acid present in soil samples during PCR amplification (9). In this study, biotin-labeled DNA probes specific for VT genes were used to capture the VTEC target DNA. The hybrids were isolated on streptavidin-coated magnetic beads, and the captured target was subsequently amplified directly by conventional PCR.

	MATERIALS AND METHODS

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Bacterial cultures. VTEC reference cultures H19, E32511, 933W, 412, and H.I.8 and non-VTEC isolates were from the laboratory collection of the Food Science Department, University of Guelph. Other VTEC isolates were obtained either from the Health of Animals Laboratory, Health Canada, or from our laboratory collection. The bacterial cultures used in this study are detailed in Table 1.

Synthesis of biotin-labeled capture probes. A mixture (1:1) of two biotin-labeled DNA probes, each specific for a particular region of the genes for VT1 or VT2, respectively, was used in the capture of target DNA fragments. The probes were synthesized by conventional PCR with primers with biotin labeling at the 5' end (Mobix, MacMaster University, Hamilton, Ontario, Canada). The primers used in the synthesis of the VT1 and VT2 capture probes are shown in Table 2. Chromosomal DNAs from E. coli H19 and E32511 served as templates for the synthesis of the capture probes for VT1 and VT2, respectively. The templates were heated at 94°C for 5 min and subsequently amplified for 30 cycles, each consisting of 94°C for 2 min, 55°C for 1 min, and 72°C for 1 min, with a model 480 DNA Thermal Cycler (Perkin-Elmer Cetus, Emeryville, Calif.). Taq DNA polymerase was purchased from Boehringer Mannheim (Laval, Quebec, Canada).

Preparation of target DNA from bacterial cultures. One milliliter of bacterial culture in brain heart infusion (BHI) broth (Difco) was centrifuged at 12,000 × g for 2 min with a bench top centrifuge (model 5415C; Brinkmann Instruments, Inc., Westbury, N.Y.). The bacterial cells obtained were resuspended in distilled water (100 µl) and boiled for 10 min. The centrifugation procedure was repeated, and the supernatant was collected for MCH-PCR.

MCH-PCR. The supernatant obtained as described above was placed in an Eppendorf tube, and a mixture of capture probes for VT1 and VT2 (1:1) was added. The mixture was boiled for 10 min and cooled rapidly on ice. Hybridization was performed at 42°C for 4 h with rotation in hybridization buffer (300 µl) containing 50% (vol/vol) formamide, 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 2% (wt/vol) blocking agent (Boehringer Mannheim), 0.1% (wt/vol) N-lauroylsarcosine, and 0.02% (wt/vol) sodium dodecyl sulfate (SDS).

Southern hybridization. Amplified PCR products were separated on 1% agarose in TBE buffer. After electrophoresis, gels were treated with 0.25 M HCl for 10 min. After a 30-min denaturation treatment in 0.5 M NaOH and 0.5 M NaCl, the gels were neutralized for 45 min in 1 M Tris and 0.6 M NaCl. The DNA was transferred to a positively charged nylon membrane (0.45-µm pore diameter; Boehringer Mannheim) by the method of Southern (28). The membrane was UV (302 nm) cross-linked for 4 min, and prehybridization was done at 37°C for 4 h with gentle shaking. Hybridization was performed at 42°C with overnight incubation according to the method of Maniatis et al. (15).

Slot hybridization. Total cellular DNA of VTEC and non-VTEC isolates was blotted on a positively charged nylon membrane (0.45-µm pore diameter; Boehringer Mannheim), air dried, and subsequently UV (302 nm) cross-linked for 4 min. Prehybridization and hybridization were both performed at 42°C with rotation in the hybridization buffer as described above. Dig-labeled capture probes were used as detection probes to identify the target. After hybridization, the membrane was treated in the way described above.

Detection limit. A subculture of E. coli O157:H7 920321 (a VT1 and VT2 producer) grown in BHI broth was serially diluted in BHI broth and incubated at 37°C with shaking for 5, 7, and 10 h, respectively. A volume of 1 ml was taken from the incubated samples and used for the preparation of target DNA and MCH-PCR amplification. Standard plate counts were conducted to determine the number of E. coli O157:H7 cells in the diluted cultures prior to incubation.

Application in food. Ground beef was purchased from a retail outlet in Guelph, Ontario, Canada. Samples of ground beef (25 g) were contaminated with E. coli O157:H7 920321 at a rate of 10⁰ to 10³ CFU/g of ground beef and incubated in BHI broth (225 ml). After enrichment at 37°C for 15 h, the culture (1 ml) was taken and centrifuged at 12,000 × g for 2 min with a bench top centrifuge. The pellets were resuspended and washed twice with distilled water (1 ml). The final suspension (100 µl) was used in sample preparation and MCH-PCR as described above.

Correlation between MCH-PCR and VT ELISA. Overnight BHI broth cultures of VTEC were centrifuged at 12,000 × g to pellet the bacteria. The supernatants were harvested and filtered through 0.2-µm-pore-diameter syringe filters, and the resulting filtrates were tested undiluted in a VT enzyme-linked immunosorbent assay (ELISA). The ELISA was performed in microtiter plates coated with 0.2 µg of the immunoglobulin G fraction of rabbit antiserum to VT1 and VT2 per well and blocked with 1% gelatin in phosphate-buffered saline (PBS). Filtrates of the VTEC cultures (100 µl) were added to two sets of duplicate wells and allowed to react at room temperature for 30 min. The wells were washed three times with PBS-0.1% Tween 20 (PBS-Tween), and each duplicate set of wells was probed separately with monoclonal antibodies to either VT1 or VT2 in PBS and incubated at room temperature for 30 min. The wells were washed three times with PBS-Tween, and 100 µl of peroxidase-labeled rabbit anti-mouse immunoglobulin G diluted in PBS was added to each well. After 30 min, the wells were washed five times with PBS-Tween, and 100 µl of tetramethylbenzidine substrate was added to the wells. The reaction was stopped after 10 min by addition of 50 µl of 0.2 M sulfuric acid, and the wells were read in a microplate reader at a wavelength of 450 nm. Negative control culture supernatants from a VT-negative strain of E. coli were included on the same assay plate, and a cutoff optical density (OD) was determined as 2× the negative control reading. Positive results were considered to be those test ODs that exceeded this cutoff OD.

	RESULTS

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Specificity of VT capture probes. Southern hybridization of PCR products (Fig. 1) with Dig-labeled capture probes confirmed their specificity. Among the VTEC reference cultures tested, the VT1 capture probe hybridized with the PCR product amplified from E. coli H19, a VT1 producer, but not with those from VT2-producing isolates E. coli 933W and E32511 (Fig. 1C), whereas the probe of VT2 hybridized only with the PCR products of two VT2 producers (Fig. 1B). PCR products were not visualized from the two E. coli strains which produce VT2 variants, 412 (a VTe producer) and H.I.8 (a VT2vha producer) (Fig. 1A). No visible signals were observed from these two isolates in Southern hybridization (Fig. 1A and B).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Specificity of capture probes as determined by Southern hybridization of PCR-amplified products from VTEC reference cultures with Dig-dUTP-labeled capture probes. Conventional PCR products were amplified from total cellular DNA of E. coli H19 (VT1), E32511 (VT2), 933W (VT2), 412 (VTe), and H.I.8 (VT2vha). After being separated by electrophoresis, the amplified products were transferred to a positively charged nylon membrane and hybridized with Dig-dUTP-labeled VT2 capture probe. Formed hybrids were detected with the Bioluminescence Detection Kit from Boehringer Mannheim. Next the probe was washed off and then rehybridized with Dig-dUTP-labeled VT1 capture probe. (A) Agarose gel containing amplified PCR products. (B) Southern hybridization with capture probe of VT2. (C) Southern hybridization with capture probe of VT1. Lanes: a, 1-kb DNA ladder; b, E. coli H19; c, E. coli E32511; d, E. coli 933W; e, E. coli 412; f, E. coli H.I.8.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Specificity of capture probes as determined by slot hybridization of total cellular DNA of 5 VTEC reference and 13 negative control cultures with Dig-dUTP-labeled capture probes. (a) Hybridization with VT1 capture probe. (b) Hybridization with VT2 capture probe. The DNA samples on each slot were as follows: A1, Salmonella typhimurium; A2, Salmonella heidelberg; A3, Salmonella hadar; A4, Salmonella enteritidis; A5, Salmonella infantis; A6, Shigella dysenteriae; B1, Aeromonas sobria; B2, Enterobacter aerogenes; B3, Serratia marcescens; B4, Klebsiella pneumoniae; B5, Escherichia coli ATCC 10789; B6, Proteus vulgaris; C1, Yersinia enterocolitica; C2 to C6, blank; D1, E. coli H19 (VT1); D2, E. coli E32511 (VT2); D3, E. coli 933W (VT2); D4, E. coli 412 (VTe); D5, E. coli H.I.8 (VT2vha); D6, blank.

MCH-PCR and correlation with the VT ELISA. With MCH-PCR, VTEC strains which produced VT1 had a 130-bp specific product (Fig. 3, lane c), and those producing VT2 yielded a product with a size of 346 bp (lane d), whereas the isolates that produced both toxins had two specific amplified PCR products (lane b).

View larger version (63K): [in this window] [in a new window]   FIG. 3.   MCH-PCR products amplified from different VT-producing E. coli O157:H7 isolates. Lanes: a and f, 1-kb DNA ladder; b, MCH-PCR products amplified from E. coli O157:H7 920321, which produced both VT1 and VT2; c, MCH-PCR product from E. coli O157:H7 920160, a VT1 producer; d, MCH-PCR products amplified from E. coli O157:H7 920191, a VT2 producer; e, negative control.

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Detection of VTEC by MCH-PCR and correlation between MCH-PCR and VT ELISA results. (A) PCR products amplified by MCH-PCR. (B) Results of VT ELISA. Amplified products were as follows (by lane): a, E. coli O115:H18; b, E. coli O121:H7; c, E. coli O157:H7 930086; d, E. coli O157:H7 920333; e, E. coli O157:H7 phage type (PT) 23; f, E. coli O157:H7 PT30; g, E. coli O157:H7 PT34; h, E. coli O157:H7 PT23; i, E. coli O157:H7 920027; j, E. coli O6:H34; k, E. coli O103:H2; l, E. coli O157:H7 PT14; m, 1-kb DNA ladder.

Detection limits. With a 5-h enrichment, MCH-PCR products were visualized from the dilution containing 10³ E. coli O157:H7 cells per ml of bacterial culture before incubation (Fig. 5A, lane g). When incubation was extended to 7 h, the detection limit reached 10² CFU/ml (Fig. 5B, lane d). The bacterial dilution containing 10⁰ CFU of E. coli O157:H7 before incubation tested positive by MCH-PCR when the samples were enriched at 37°C for 10 h (Fig. 5C, lane e). Southern hybridization of MCH-PCR products did not lower the detection limit (data not shown).

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Detection limit in bacterial culture. E. coli O157:H7 920321 was inoculated into BHI broth and incubated at 37°C until the OD600 reached 0.95. The culture was serially diluted in BHI broth to concentrations between 108 and 100. The diluted cultures were incubated at 37°C for 5, 7, and 10 h. A volume of 1 ml was taken from each dilution and used to prepare the DNA template and in the MCH-PCR. (A) Agarose gel containing MCH-PCR products amplified from the samples incubated at 37°C for 5 h. Lanes: a and k, 1-kb DNA ladder; b to j, PCR products amplified from bacterial cultures containing 108 to 100 CFU of E. coli/ml. (B) Agarose gel containing MCH-PCR products amplified from samples incubated at 37°C for 7 h. Lanes: a and f, 1-kb DNA ladder; b to e, MCH-PCR products amplified from bacterial cultures containing 104 to 101 CFU of E. coli/ml. (C) Agarose gel containing MCH-PCR products amplified from samples incubated at 37°C for 10 h. Lanes: a and f, 1-kb DNA ladder; b to e, PCR products amplified from bacterial cultures containing 103 to 100 CFU of E. coli/ml.

Detection of VTEC in ground beef. With a 15-h incubation, ground beef samples artificially contaminated with E. coli O157:H7 920321 at a rate of 10³, 10², 10¹, or 10⁰ CFU/g of ground beef all tested positive by MCH-PCR (Fig. 6).

View larger version (77K): [in this window] [in a new window]   FIG. 6.   Detection of VTEC from artificially contaminated ground beef. Samples of 25 g of ground beef were contaminated with E. coli O157:H7 920321 at a rate of 103 to 100 CFU/g. The contaminated meat samples were preenriched in 225 ml of BHI broth at 37°C for 15 h. A volume of 1 ml was taken from each sample and used in MCH-PCR amplification. Lanes: a and g, 1-kb DNA ladder; b to e, PCR products amplified from ground beef initially contaminated with 103, 102, 101, and 100 CFU of E. coli/g of ground beef, respectively; f, negative control (uninoculated meat).

	DISCUSSION

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Use of conventional PCR in the detection of VT genes from VTEC isolates has been reported by Pollard et al. (21), Lin et al. (14), and Thomas et al. (30). These authors all found that the technique was both sensitive and specific and may be useful for rapidly screening clinical specimens for VTEC. Gannon et al. (4) and Witham et al. (31) have reported the PCR detection of VTEC in ground beef, both using purified bacterial DNA.

MCH-PCR was used successfully in the detection of Pseudomonas fluorescens cells labeled with lux genes in the presence of humic acid, a PCR inhibitor commonly present in soil samples (9). Since food components usually exert a similar inhibition towards the efficiency of DNA amplification, attempts were made to apply the MCH-PCR technique to the amplification of the genes that encode VTs. This study demonstrates that the technique is a sensitive and specific method for the detection of pathogens from foods without the need for isolation and purification of template DNA. With nonselective enrichment, the detection limit was as low as 10⁰ CFU/g of ground beef (Fig. 6) or 10³, 10², or 10⁰ CFU/ml in pure culture, depending on the length of enrichment (Fig. 5). The detection limits were repeatable but accomplished by including an enrichment step. If the enrichment step was excluded, at least 10⁵ or 10⁶ CFU/ml or CFU/g was required to achieve a positive detection. This was probably because of the small sample size (1 ml) and elimination of colony isolation (from ground beef) and purification of bacterial DNA.

Two VT2 variant-producing isolates were included in the detection: E. coli H.I.8 was isolated from an infant with diarrhea (5), and 412 was isolated from a pig with edema disease (6). The genes that encode VT2 variants, of both human and porcine origin, were not amplified by MCH-PCR. Therefore, other than VTEC detection, the same method can also be used to differentiate VT2- from VT2 variant-producing E. coli isolates. Additional probes and primers (14) could be incorporated into the procedure if the detection of VTs and their variants is desired.

The VT1 primers used for PCR targeted a 130-bp fragment (Fig. 3, lane c) in the region coding for the B subunit of the toxin and VT2 primers amplified a 346-bp fragment (Fig. 3, lane d) coding for the A subunit of the toxin (21). During MCH-PCR, the genes for VT2 were sometimes amplified less efficiently than those for VT1 (Fig. 5C, lane e). It is not clear whether this phenomenon is associated with the difference in the size of the amplified PCR products.

The amount of the beads used in MCH-PCR amplification was found to be critical. An excessive amount of beads present in the PCR mix during the MCH-PCR could cause the failure of the amplification. It was unclear whether the beads inhibited the polymerase enzyme or whether the lack of amplification was due to some other cause. At the initial stage of the research, a different approach was attempted to release the DNA hybrid, after the hybridization, from streptavidin-coated beads with 6 M urea for protein denaturation and 100% ethanol for DNA precipitation. The results were discouraging (not shown), and this was probably due to the introduction of additional chemicals into the DNA samples.

A 100% correlation between MCH-PCR and the VT ELISA was obtained. One of the VTEC isolates tested, E. coli O115:H18, had an anti-VT2 OD of >3.000, indicating that the isolate produced VT2. Its anti-VT1 OD was 0.159, greater than 2× the negative control reading (0.144) (Fig. 4B); therefore, it was a weak VT1 producer. The results from MCH-PCR demonstrated that the isolate carried the genes for both VT1 and VT2 (Fig. 4A, lane a). The low reading from the immunoassay could be explained as a specific phenotype characterized by a low level of VT1 production caused by possible genetic alteration of the nucleotide sequence that encodes the VTs and their regulatory elements. This example also demonstrates the advantage of the MCH-PCR method for the detection of potential VT1 producers that express only small quantities of the toxin under the culture conditions used in the immunoassay.

Because E. coli O157:H7 has been declared an adulterant in beef by the U.S. Department of Agriculture, it is important that tests are capable of detecting the organism at low concentrations. Although E. coli O157:H7 is the main serotype implicated in human illness, other VTEC strains have been shown to be human pathogens, and there is considerable debate over whether assays for O157:H7 alone or all VTEC strains should be employed for food testing. If an O157:H7-specific test was required, the MCH-PCR could be multiplexed with probes specific for that serotype. The results in this study demonstrated that MCH-PCR was capable of detecting low initial numbers of VTEC in ground beef following enrichment without the need for complicated DNA isolation and purification steps. The technique could also be automated, thus making it of use for routine screening of food samples.

The results obtained from the present research are encouraging. Possible future investigations will include the use of the technique for the detection of multiple food-borne bacterial pathogens, such as VT- and enterotoxin-producing E. coli, Salmonella spp., and Yersinia enterocolitica.