Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli

The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes. Systems designed for the detection of genes of the stx(1), type did not detect any variant genes of the stx,type and conversely, no stx(2) type-specific systems detected stx(1), variant genes. Among five stx(2) type-specific systems, none detected the stx(2ev) gene, and two detected the stx(2e) gene. Among systems designed for screening genes of the both stx(1) and stx(2), types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains. Shiga-toxin-producing E. coli frequently carry more than one stx variant gene. Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size. The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx(1), stx(2), stx(2c), stx(2e) and stx(2ev). Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.