Detection and quantification of Shiga toxin-encoding genes in sheep faeces by real-time PCR

Sheep faeces may be an important source of Shiga toxin (Stx)-producing Escherichia coli. We have, therefore, established and evaluated a real-time 5′-nuclease PCR assay to quantify the stx1 and stx2 genes in sheep faeces. The detection limit of our assay for both stx1 and stx2 in spiked samples corresponded to 102-103 CFU/g, which is lower than for other assays for measuring these genes in faecal samples. Quantification values for our assay ranged from 102 to 107 CFU/g faeces. The assay was evaluated on native, unspiked faeces. All sheep tested (n=7) shed stx1, and the quantitative results corresponded to the gene copies in 103-104 CFU/g. The level of stx2, however, was below the quantitative detection limit in all the samples analyzed. This quantitative stx1 and stx2 assay may be important in assessing whether sheep harbouring Shiga toxin-producing bacteria represent a potential hazard to human health.