Single-step polymerase chain reaction for combined gene detection and epidemiological typing in three bacterial...

We describe a new polymerase chain reaction (PCR) for combined gene detection and epidemiological typing (COGEDET), which allows bacterial typing and gene detection in a one-step assay. This assay, in which target gene-specific primers are used under low-stringency annealing conditions, was evaluated on 32 Staphylococcus aureus strains using toxic shock syndrome toxin 1 (tst) primers, 30 Clostridium difficile strains using toxin A (toxA) primers, and 30 Escherichia coli strains using cytotoxic necrotizing factor (cnf) primers. Typing performances with COGEDET were compared to those of conventional random amplification polymorphic DNA (RAPD), and gene detection performances, to those of conventional PCR followed by Southern blot hybridization. Concordances between conventional PCR/Southern blot and COGEDET were 96.9, 100 and 96.7% for the detection of the tst, toxA and cnf genes, respectively. Discriminatory indexes for the conventional RAPD and COGEDET techniques were similar in the three bacterial species tested. These results show that the COGEDET assay can replace two separate assays for typing and genes detection respectively, thus saving both technicians' time and reagents.