Use of gene amplification to detect Clostridium difficile in clinical specimens

The combined use of an enrichment broth and gene amplification following simple DNA extraction to detect toxigenic strains of Clostridium difficile in feces was investigated by examining feces from 329 cases of suspected C. difficile infection. DNA was extracted by heating the washed centrifuged deposit from the broth in a microwave oven. For comparison, specimens were tested concurrently using standard methods for culture and cytotoxin testing. Amplified fragments were identified by molecular weight estimation, restriction enzyme digestion patterns and Southern blot hybridization. The combination of an enrichment broth followed by gene amplification was shown to be a sensitive, specific and rapid method for detecting toxigenic strains of C. difficile in feces. Use of the method in diagnostic laboratories may require the development of improved detection and verification systems for the amplified gene fragment.