Cloth-based hybridization array system for the identification of Escherichia coli O157:H7

Abstract

A simple cloth-based hybridization array system (CHAS) utilizing polyester cloth as the solid phase for DNA hybridizations has been adapted as a tool for the identification of presumptive verotoxigenic Escherichia coli O157:H7 colonies isolated from foods by standard culture techniques. Key indicator genes for this organism, rfbO157, fliCH7, vt1 and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes, respectively) were amplified in a multiplex PCR incorporating digoxigenin-dUTP, followed by hybridization of the amplicons with an array of specific oligonucleotide probes immobilized on polyester cloth, and subsequent immunoenzymatic assay of the bound digoxigenin label. This system includes a simple internal amplification control (IAC) to gauge PCR inhibition based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth. The CHAS provided sensitive and specific detection of the E. coli O157:H7gene markers, exhibiting the expected patterns of reactivity with a panel of various target and non-target organisms.