Roche Applied Science has revealed that its Magna Pure sample preparation system and LightcyclerO systems are being used for high-throughput examination of EHEC pathogens in primary stool samples.
According to the company, there has been an increase in the number of cases of bloody diarrhoea associated with haemolytic-uremic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC).
To streamline the workflow for detecting the EHEC bacterium, the automated Magna Pure sample preparation system for nucleic acid isolation and LightcyclerO real-time PCR instrument are being used.
In a recent study (in preparation), human stool samples were processed using the high-throughput Magna Pure 96 workstation, isolating high-quality total genomic DNA from primary faecal specimens.
Targeting the toxin genes of EHEC by a LightcyclerO real-time PCR test demonstrated that faecal specimens can be used as starting material to detect EHEC infection in epidemic situations reducing the turnaround from at least overnight incubation to hours.
The fully automated robotic Magna Pure 96 instrument used in this study isolates nucleic acids from 96 samples in approximately one hour.
The Roche platform is an efficient, cost-effective way to prepare template DNA from large numbers of stool samples in epidemic situations.
Subsequent LightcyclerO 480 real-time PCR was found to be accurate and reproducible using proven specific, sensitive PCR primers and probes, targeting the Shiga toxin genes of EHEC, available as LightmixO EHEC.
To ensure the robustness of the entire Magna Pure 96 and LightcyclerO workflow for EHEC testing, a pathogen universal protocol was established using a 200ul sample volume and 100ul elution volume.
E.coli Shiga toxin genes were amplified in the presence of two pairs of fluorescently labelled hybridisation probes targeting stx-1 and stx-2, followed by LightcyclerO 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.
Systematic testing of this workflow should significantly contribute to the early detection and surveillance of future EHEC infections, as well as other emerging bacterial epidemics.
The team has evaluated a novel four-colour multiplex assay using hydrolysis probes for the LightcyclerO 480 instrument (publication submitted), targeting both toxin genes (stx1 and stx2), the wzx gene (marker for the O104 serotype), and an internal control.
This assay enables the analysis of suspected samples in a single reaction, for a faster more cost-effective time-to-result, which is particularly important during an outbreak, when many samples have to be studied in short time.