Yutaka Takarada

Evaluation of Enzyme-Labeled Oligonucleotide Probes to Identify Enterohaemorrhagic Escherichia coli

Alkaline phosphatase‐conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.

Development of an Enzyme‐Labeled Oligonucleotide Probe for Detecting the Escherichia coli Attaching and Effacing A Gene

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) can produce attaching and effacing (AE) lesions on intestinal epithelium in vitro and in vivo. A gene necessary to cause the AE lesion has been identified and designated Escherichia coli attaching and effacing A (eaeA) gene. In this study, an alkaline phosphatase (ALP)‐conjugated oligonucleotide probe for the eaeA gene was developed and used to detect the eaeA gene among 163 strains of classical EPEC and 25 strains of EHEC O157. The prevalence rates of eaeA gene in the strains of classical EPEC and EHEC O157 were 51.5 and 100%, respectively. The eaeA‐positive rate (60.0%) in strains of class I EPEC serogroups (026, 055, 086, O111, O119, O125, O126, O127, O128ab, and O142) was significantly higher than that (22.9%) in strains of the class II EPEC serogroups (O18, O44, O114) (P<0.01). A total of 109 eaeA‐positive classical EPEC and EHEC O157 were positive for fluorescent actin staining (FAS) assay, whereas 79 eaeA‐negative classical EPEC were negative. Both the sensitivity and specificity of the eaeA probe versus the FAS assay positivity were 100%. Thus, use of the ALP‐conjugated oligonucleotide probe for the eaeA gene would be specific and reliable in identifying the adherence capability of EPEC and EHEC.