Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR
Introduction
Enterohemorrhagic Escherichia coli (EHEC), including E. coli O157:H7, comprise a subset of Shiga toxin-producing E. coli (STEC). EHEC are predominantly associated with hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans (Karch et al., 1996, Kaper and O'Brien, 1998, Ibekwe et al., 2002). EHEC O157:H7 is implicated in the vast majority of outbreaks and sporadic cases of bloody diarrhea compared to other EHEC serotypes. The pathogenicity of E. coli O157:H7 is associated with several virulence factors, including Shiga toxins 1 and 2 (encoded by genes stx1 and stx2), and intimin (encoded by the eaeA gene). Shiga toxins cause hemorrhagic colitis and HUS through cytopathic effects on vascular endothelial cells of kidneys, intestines, the central nervous system and other organs. Intimin facilitates adherence to intestinal villi, forming the attaching-and-effacing intestinal lesion (Brogden et al., 2000).
The sources of EHEC infections include raw ground beef, unpasteurized milk, untreated water and apple cider (Oberst et al., 1998, Pyle et al., 1999). Most disease outbreaks have involved foods of bovine origin such as beef and raw milk because cattle are a major reservoir for E. coli O157:H7 and many other EHEC (Siragusa et al., 1999, Ogden et al., 2000). Undercooked ground beef has been linked to most of the E. coli O157:H7 outbreaks.
Established methods for identifying E. coli O157:H7 have been directed at immunological or genetic targets. Antigenic targets have included the E. coli somatic (O157) or flagellar (H7) antigens and the virulence-associated Shiga toxins 1 and 2. PCR-based detection procedures used to identify E. coli O157:H7 have targeted a number of genes including stx1 and stx2 genes and eaeA (Meng et al., 1996, Stewart et al., 1998, Uyttendaele et al., 1999). However, conventional PCR-based detection methods have not been sufficiently sensitive to detect E. coli O157:H7 without an enrichment culture step. A more rapid method would improve the probability of identifying the source and preventing the further spread of infection. Immunomagnetic separation (IMS), followed by culture or other identification methods, has been used to capture cells from enrichment culture by magnetic particles coated with antibodies against the surface antigens of E. coli O157:H7 (Wright et al., 1994, Bennett et al., 1996, Pyle et al., 1999, Barkocy-Gallagher et al., 2002, Sun et al., 2002). A method combining IMS and a DNA microarray has been developed to detect E. coli O157:H7 from poultry carcass rinsate without the need for an enrichment step (Chandler et al., 2001). However, DNA microarrays are currently comparatively expensive and technically complex for screening by the food industry.
The objective of this research was to develop a rapid, specific and quantitative method to detect E. coli O157:H7 in ground beef. E. coli O157:H7 may be present in ground beef at very low levels (<100 CFU/g), while the level of other organisms may be as high as 104–106 CFU/g (Tsai et al., 2000). Our method combined IMS for cell capture and concentration with real-time (5′-nuclease) PCR for detection and quantification of IMS-captured E. coli O157:H7.
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Bacterial cultures
E. coli O157:H7 (ATCC 51657), E. coli O26 (ATCC 11840) and E. coli FDA strain Seattle 1946 (ATCC 25922) were purchased from ATCC (American Type Culture Collection, Manassas, VA). E. coli O157:H7 was grown in tryptic soy broth (TSB) (BD, Sparks, MD) with 0.5% yeast extract (Difco, Detroit, MI); E. coli O26 was grown in nutrient broth (Difco, Detroit, MI); the FDA strain of E. coli was cultivated in tryptic soy broth. All test organisms were cultured at 37 °C. For experiments requiring a
Cell capture efficiency
Preliminary experiments were performed to optimize the coverage of antibody on Pro-A beads, the incubation time and elution buffer composition for the most efficient cell capture. Quantitative data were obtained by plate counts. When a cell suspension of about 104 cells/ml in PBS was incubated with approximately 107 Pro-A beads and 1012 copies of Anti-O157 antibody for 2 h on ice, the highest percentage of cells captured by the beads was approximately 50% (Fig. 2). This result was also
Discussion
This study demonstrates the potential for combining IMS with real-time PCR to create a rapid method for detecting E. coil O157:H7. Such a rapid assay could allow one to determine the source of an outbreak quickly or to prevent disease altogether. It is also specific and quantitative. There was no cross-reactivity between E. coli O157:H7 and the other strains of E. coli that were tested, although there was overestimation of the abundance when E. coli O157:H7 was mixed with a large number of
Acknowledgements
This research was supported by the Office of Naval Research (Award number N00014-00-1-0699). We thank Cynthia Bruckner-Lea, Darrell Chandler and Susan Cordova for helpful input.
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