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 stx(1) and stx(2) genes in sheep faeces. The detection limit of our assay for both stx(1) and stx(2) in spiked samples corresponded to 10(2)--10(3)CFU/g, which is lower than for other assays for measuring these genes in faecal samples. Quantification values for our assay ranged from 10(2) to 10(7)CFU/g faeces. The assay was evaluated on native, un-spiked faeces. All sheep tested (n=7) shed stx(1), and the quantitative results corresponded to the gene copies in 10(3)--10(4)CFU/g. The level of stx(2), however, was below the quantitative detection limit in all the samples analyzed. This quantitative stx(1) and stx(2) assay may be important in assessing whether sheep harbouring Shiga toxin-producing bacteria represent a potential hazard to human health.