Assay Solution for Rapid EHEC Identification
Product News May 31, 2011
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, 329 laboratory confirmed cases have been reported, three of them with fatal outcome (Robert Koch Institute’s report May 29th). The majority of cases are in northern Germany, but recently emerging cases from southern Germany and other European countries have also been reported. The German Consiliary Center for EHEC has identified EHEC producing Shiga toxin 2 (Stx2), as the causative agents. Possible sources are reported to be contaminated vegetables.
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.