Detection of shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP

Abstract

A technique has been developed for the detection of Shiga toxin- and Shiga-like toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli.