LightCycler^® Real-Time PCR Systems for EHEC identification

With the recent outbreak in Europe of Enterohaemorrhagic Escherichia coli (EHEC), Roche Applied Science  and TIB Molbiol are providing scientists with complementary technologies that can be used for the rapid identification and detection of EHEC.

As of the second week of May 2011, there has been a significant increase in the number of cases of bloody diarrhea associated with hemolytic-uremic syndrome (HUS). Currently, 3304 cases of EHEC or HUS cases had been reported to the Robert-Koch-Institut, 38 of them with fatal outcome (Robert Koch Institute’s report June 16^th

Fast, reliable detection of the EHEC bacterium is essential for monitoring and controlling EHEC infections. The most appropriate targets for the rapid, sensitive identification of the EHEC bacterium are unique genetic sequences, which can be amplified and detected using the polymerase chain reaction (PCR) method. Roche Applied Science provides complete systems for PCR, including LightCycler^®Instruments, software, reagents and consumables. These detection systems are designed for life science research to enable the rapid development and adaptation of PCR assays, accelerating the progress of appropriate diagnostic tools.

The detection of EHEC by real-time PCR using the LightCycler^® platform was already developed in 2002, and published jointly (J. Clin. Microbiol. (2002) 40, 2555-2565) by scientists from the universities of Regensburg (Prof. Dr. U. Reischl), Muenster (Prof. Dr. H. Karch), and the CDC in Atlanta (Dr. N. Strockbine). Since 2002, this real-time PCR test has been widely adopted by laboratories worldwide.

The new LightMix^® Kit EHEC from TIB Molbiol contains pre-mixed primers and probes and an appropriate positive control for use with the LightCycler^® Instruments. The test detects and differentiates both Shiga toxin genes and can be performed in less than one hour. The kit is based on the previously published assay from Grys et al., (J. Clin. Microbiol. (2009) 47, 2008-2012).

The standard analytical procedure for EHEC detection requires an overnight bacterial culture from patient stool samples prior to performing the PCR assay. To expedite time-to-result, such as for the EHEC outbreaks and samples with bloody diarrhea, it is possible to isolate the bacterial nucleic acids directly from stool samples using automated isolation methods of the Roche Applied Science MagNA Pure Sample Preparation Systems. This automated workflow followed by direct real-time PCR is currently under evaluation by a number of laboratories.