E. coli virulence genotyping PCR

Price: $55.00

Detection of E. coli virulence factors by PCR.

PCR is performed after isolation of E. coli by culture in the Bacteriology lab.

E. coli isolates are identified by standard biochemical reactions in the bacteriology lab and submitted on MacConkey Agar or other appropriate (TSA, CBA) plates, or may be received from other labs for testing.

Feces and tissues: sealed, leak-proof plastic bag or container.

Label all samples. Store refrigerated. Ship overnight on cold packs in an insulated container.

Diarrhea in calves between the ages of one and 6 days old is commonly caused by enterotoxigenic E. coli (ETEC). Diarrhea and dysentery in older calves is sometimes caused by attaching and effacing E. coli (AEEC) and Shiga (also called shiga-like or verotoxin) toxin-producing E. coli. ETEC has both fimbrial and enterotoxin virulence factors. In cattle, F5 (K99) fimbriae, which mediate adherence to the ileum, are found in most calf ETEC. (The other common calf fimbria, F41, is almost always found in conjunction with F5.) Calf ETEC produces heat stable enterotoxin (Sta), which causes hypersecretion into the gut lumen.

A chromosomal gene, eaeA, encodes the protein intimin, which is involved in attaching and effacing activity. Enteropathogenic E. coli (EPEC) are AEEC which do not produce toxin genes. STEC produce either one or both types of Shiga toxins, Stx1 and Stx2, which act by inhibiting protein synthesis (and are toxic to cultured Vero cells). E. coli which produce both intimin and Shiga toxins are called enterohemorrhagic E. coli (EHEC) because of their association with hemorrhagic colitis in humans.

This multiplex PCR was adapted from a previously published paper, with primers shown to be specific for the F5 (K99), F41, stable toxin (Sta), Shiga toxin (Stx1 and Stx2), and eaeA (intimin) genes. These primers also detect the stx2 variant stx2e, which has mainly been found in E. coli from pigs. This assay is used primarily for bovine samples, but may be used for other species, or to confirm the presence of stx1 or stx2 and intimin in E. coli 0157:H7 suspect isolates. A separate multiplex PCR assay is done for pig isolates in order to detect F4 (K88), ST1B, and labile toxin (LT).

The assay is performed on cultured E. coli samples from the Bacteriology lab.