![]() 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).
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