Detection of verotoxigenic Escherichia coli in bull semen using the polymerase chain reaction.

Oligonucleotide primers used in a polymerase chain reaction (PCR) protocol detected the verotoxin 2 (VT2) gene in E. coli present in experimentally contaminated bull semen. The VT2 (Shiga-like toxin II [SLT-II]) primers targeted a 346-bp fragment of the gene coding for the A subunit of the toxin. PCR products, corresponding to the VT2 gene sequence, were amplified from template E. coli nucleic acid extracted from 18-h broth culture and from E. coli in contaminated semen in the undiluted state, diluted in egg yolk-Tris and diluted in milk. The sensitivity of the assay to detect E. coli was determined to be 1 pg of nucleic acid, and as few as 10-20 E. coli organisms/ml could be detected in raw and diluted semen. Preliminary confirmation of the PCR product was accomplished by slot blot hybridization to a radiolabeled specific oligoprobe. Sequencing of the PCR products identifying VT2 gene sequence revealed 99.7% homology with published gene sequences for VT2. This study demonstrates the feasibility of applying PCR technology for the detection of E. coli in bovine semen. This technique may find wide application for the detection of other pathogens that may be present in semen.