Establishing a real-time PCR method for analysis of stx2 expression

Abstract

STEC (Shiga toxin-producing E.coli) are important human pathogens, which can potentially cause hemolytic uremic syndrome (HUS). An important predictor of serious disease is the production of the variant Shiga toxin 2 (Stx2). This thesis is part of a larger project that aims to establish a method forquantitation of stx2 in the form of a real-time PCR assay. We conducted experiments to find optimal culture conditions, performed DNA extraction and validation of PCR results. Furthermore, we have optimized the RT-PCR for both stx2 and the housekeeping gene tufA, and designed standard curves for calculating the efficiency of both of these. In this project we have produced experimental results which in the future can be used in the further development of an RT-PCR assay for stx2.