Comparison of the partial 16S rRNA gene sequences and development of oligonucleotide probes for the detection ofEscherichia coli cells in water and milk

Abstract

The time and accuracy limitations of the commonly used methods for Escherichia coli detection in food and water suggest the need for rapid and specific methods. DNA hybridization is one of these methods. To develop E. coli -specific DNA probes, the primary sequences of the V3 and V6 regions of the 16S rRNA genes of non-pathogenic and pathogenic E. coli strains, were determined and compared with those of the non- E. coli strains, including strains of the family of Enterobacteriaceae. Two oligonucleotides named as 16El and EV6 were then designed from the sequence unique to the E. coli cells. They were P32-labelled and tested for their colony-hybridization specificities to the non-pathogenic and pathogenic E. coli strains. Results showed that all the E. coli strains tested generated positive hybridization signals, while none of the non- E. coli strains tested gave a false positive result (except forShigella spp.) When these probes were used for the detection of E. coli cells in contaminated tap-water and milk samples it was found that as low as 100cfu 100 ml−1of tap-water or ml−1milk could be detected if an 8-h preculture step was performed prior to the hybridization assay.