A nested PCR followed by magnetic separation of amplified fragments for detection of Escherichia coli Shiga-like toxin genes.

The Shiga-like toxin (SLT) I and II genes in cytotoxic Escherichia coli strains were detected using a polymerase chain reaction (PCR) procedure. Identification and differentiation of SLT I and II was carried out using primers giving PCR-generated DNA fragments of different size for the two cytotoxins. A two-step PCR procedure utilizing three primers in a nested configuration for both SLT I and II was combined with magnetic separation to identify the toxin genes in a rapid, specific and sensitive test system designated DIANA (Detection of Immobilized Amplified Nucleic Acid). The first PCR was carried out using standard methods, and the product generated was used as primer in the second PCR. In this procedure one of the primers from the first PCR was used with biotin label, and the second (inner) primer was 32P-labelled. The double-stranded DNA fragments generated containing the two primers, were biotinylated on one 5' end and 32P-labelled on the other 5' end. These fragments were separated from the solution using streptavidin-coated super-paramagnetic microscopic beads. The test could detect and differentiate between SLT I and II in a positive/negative ratio of more than 20. The assay could detect five SLT-positive E. coli organisms in the 5 microliters test sample. The presence of 100-fold more SLT-negative strains in a sample did not adversely affect the test signal.