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

Abstract

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.

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

“…For Southern blot analysis, the DNAs were transferred to a Zeta-Probe GT Blotting Membrane (Bio-Rad Laboratories) and were hybridized with alkaline phosphatase-conjugated oligonucleotide probes that are specific for stx 1 and stx 2 genes (VT1 probe and VT2 probe; Toyobo, Shiga, Japan). The selected nucleotide base sequences for the probes and detection procedures were described by Yoh et al [18]. …”

Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates

“…The digested DNAs were separated by conventional agarose 0.8% gel electrophoresis, transferred by Hybond-N membrane (Amersham, Buckinghamshire) and probed with alkaline phosphataselabelled probes (Toyobo, Tokyo, Japan), following the manufacturer's instructions. Probes for the A subunits of stx 1 and stx 2 comprised DNA corresponding to the nucleotide base sequences from 1106 to 1133 and from 1099 to 1127 from the initiation codon of the stx 1 and stx 2 genes, respectively [26]. In the stx 2 gene, there is only one Sma I restriction site, located at position 302± 307 from the initiation codon of the gene [5,20].…”

“…Southern blot hybridisation of the chromosomal DNA preparations was performed to detect stx-containing fragments of phage DNA inserted in the host's DNA. The blotting was performed as described previously [25] and subsequent hybridisation was performed by the method described by Yoh et al [26]. Brie¯y, the total DNA preparations were digested by several endonucleases, including Sma I, HindIII and EcoRI (Takara).…”

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

“…A total of 163 strains of classical, non-verocytotoxin (VT) I-and IIproducing EPEC serotypes, 3 of VT-producing E. coli 0157:H -, and 22 of VT-producing E. coli 0157:H7 , assessed by DNA probes for VT I and II (25), were examined in this study. Fifty-one of the E. coli strains isolated from nondiarrheal stool specimens, and 35 strains of other enteropathogens including Salmonella spp., Shigella spp., Aeromonas spp., Citrobacter spp ., Providencia spp., Edwardsiella spp., Proteus spp., Enterobacter spp., Chromobacteria spp.…”