Detection of Shiga-Like Toxin ( <i> stx ₁ </i> and <i> stx ₂ </i> ), Intimin ( <i>eaeA</i> ), and Enterohemorrhagic <i>Escherichia coli</i> (EHEC) Hemolysin (EHEC <i>hlyA</i> ) Genes in Animal Feces by Multiplex PCR

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.