Amplified DNA bands were progressively lost in descending order from the largest (890-bp) eaeA prod- uct, which was lost between 37 and 18 genome equivalents, to the 165-bp EHEC hlyA product, which is detectable at less than 3 genome equivalents (Fig. The sensitivity of the EHEC multiplex PCR assay was estimated to be between 37 and 18 genome equivalents. coli O111:H Ϫ organisms per ml was determined.

A stock suspension containing 6.3 ϫ 10 7 E. coli O91:H Ϫ by the Victorian Infectious Diseases Reference Lab- oratory, was not detected by multiplex PCR. stx 2, which had previously been detected in E. The geno- typic results obtained by the multiplex EHEC PCR assay were in agreement with phenotypic data provided by the Victorian Infectious Diseases Reference Laboratory for 17 of the known 18 factors. All reference strains except O91:H Ϫ contained the EHEC hlyA gene. Three strains contained only stx 1, and the re- maining two strains contained stx 2. stx gene sequences were detected in all six reference EHEC serotypes, and one strain contained both stx 1 and stx 2.

DNA extracted from strains representative of each of six EHEC serotypes was the template for a range of PCR ampli- fication products (Fig. No amplification products were present in either the negative control (a fecal culture previously determined to lack sequences for any of the four virulence determinants by multiplex PCR) or a water control (no nucleic acid) after PCR (results not shown). coli O111:H Ϫ DNA template as a positive control (Fig. PCR products representing each of the four target EHEC virulence factors were amplified with E. Membranes were subsequently exposed to X-ray film (Kodak) at room temperature for 10 to 60 min prior to development. Bound DIG-labelled probes were detected with CSPD (Boehringer GmbH, Mann- heim, Germany) by following the manufacturer’s instructions. All washes were per- formed in a minihybridization oven.

After incubation, mem- branes were washed twice in 2 ϫ SSC (1 ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% sodium dodecyl sulfate (SDS) for 15 min at room temperature followed by two washes in 0.5 ϫ SSC–0.1% SDS at 68☌. Hybridization was carried out in a minihybridization oven (Hybaid) at 58☌ by using DIG Easy Hyb (Boehringer GmbH, Mannheim, Germany) with the de- natured DIG-labelled DNA probe. Colony hybridization was carried out with a DIG chemilumi- nescence detection kit (Hyb Boehringer Mannheim) per the manufacturer’s instructions. subse- quently used to transfer the bacteria onto nylon Hybond H ϩ membrane (Amersham, Little Chalfont, United Kingdom).

Hybaid mini hybridization oven manual