Roche Applied Science's MagNA Pure 96 Instrument and LightCycler Real-Time PCR Instrument are being used to streamline the workflow in detecting hemolytic-uremic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC) in primary stool samples in Germany, Sweden, Russia as well as other European countries.
The study was carried out by Laboratory of Dr Krause & Colleagues, Institute of Medical Microbiology and Hygiene, University Hospital of Regensburg and TIB MOLBIOL in Germany.
A new Pathogen Universal Protocol was established using a 200 µl sample volume and 100 µl elution volume, to ensure the robustness of the entire MagNA Pure 96 and LightCycler workflow for EHEC testing.
E coli Shiga toxin genes were amplified in the presence of two pairs of fluorescently labeled hybridization probes targeting stx-1 and stx-2, followed by LightCycler system melting curve analysis discriminating the toxin gene subtypes.
The bacterial parC gene (encoding the topoisomerase IV subunit of Enterobacteriaceae) was used as an extraction control to prove presence of extracted DNA and to exclude PCR inhibitors.
The team has evaluated a four-color multiplex assay using hydrolysis probes for the LightCycler 480 Instrument targeting both toxin genes (stx1 and stx2), the wzx gene (marker for the O104 serotype), and an internal control.
This new assay enables the analysis of suspected samples in a single reaction, for an effective time-to-result.