SINGLE-STEP POLYMERASE-CHAIN-REACTION FOR COMBINED GENE DETECTION ANDEPIDEMIOLOGIC TYPING IN 3 BACTERIAL MODELS

We describe a new polymerase chain reaction (PCR) for combined gene de tection and epidemiological typing (COGEDET), which allows bacterial t yping and gene detection in a one-step assay. This assay, in which tar get gene-specific primers are used under low-stringency annealing cond itions, 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 c ytotoxic necrotizing factor (cnf) primers. Typing performances with CO GEDET were compared to those of conventional random amplification poly morphic DNA (RAPD), and gene detection performances, to those of conve ntional PCR followed by Southern blot hybridization. Concordances betw een conventional PCR/Southern blot and COGEDET were 96.9, 100 and 96.7 % for the detection of the tst, toxA and cnf genes, respectively. Disc riminatory indexes for the conventional RAPD and COGEDET techniques we re similar in the three bacterial species tested. These results show t hat the COGEDET assay can replace two separate assays for typing and g enes detection respectively, thus saving both technicians' time and re agents.