Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic Yersinia and Shiga toxin-producing Escherichia coli from foods

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), ampl ification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathog enic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. T his technology allows the synthesis of a variety of shelf-stable probe reag ents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: v irF and yadA in enteropathogenic Yersinia and stx(1) and stx(2) in Shiga-li ke toxin-producing Escherichia coli. Results of DNA hybridizations of dot b lots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent wi th results of equivalent PCR analyses. DNA colony hybridization with noniso topic virF probes of colonies arising on spread plates from artificially co ntaminated food homogenates was able to detect potentially pathogenic Y. en terocolitica. When compared with oligonucleotide probes, amplicon probes ar e much less sensitive to changes in hybridization and wash temperatures, al lowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at -20 degrees C for more than 8 mont hs. This method conveniently generates probes that are safe, stable, inexpe nsive, reusable, and reliable.