Detection of shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR

Abstract

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 and stx2), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors.

FIG. 1

Sensitivity of multiplex PCR in detecting EHEC virulence factors using serial dilutions of E. coli O111:H−. DNA markers are indicated on the right (numbers are molecular weights, in base pairs). Lanes 1 through 13 contain 6,250, 2,500, 630, 372, 186, 93, 46, 37, 18, 9, 3, 1.5, and <1 genome equivalents, respectively.

FIG. 2

Multiplex PCR analysis of EHEC reference strains. Arrows A and B refer to nonspecific PCR bands (see the text). Lanes: 1, E. coli O111:H8; 2, E. coli O157:H7; 3, E. coli O128:H2; 4, E. coli O91:H−; 5, E. coli O113:H21; 6, E. coli O5:H−; 7, E. coli O111:H− positive control.

FIG. 3

Multiplex PCR analysis of bovine fecal samples. Lanes: 1, O111:H− positive control; 2 through 10, fecal samples collected on a farm previously identified as containing EHEC-positive animals.