Automated 5′ nuclease assay for detection of virulence factors in porcine Escherichia coli

This paper describes the development of a 5' nuclease assay for detection of virulence factor genes responsible for colonization factors and toxins in Escherichia coli isolated from pigs. Colonization factors were F4, F5, F6, F18, F41 and the outer membrane protein intimin. Toxins were heat stable (STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coli 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coli reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coli field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains. When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory. (C) 2001 Academic Press.