Clostridium difficile-associated disease (CDAD) is a major problem for hospitalized patients recieving antibiotics or antineoplastic agents. Current labotatory methods for diagnosing CDAD include culture and assays for detection of the toxins produced by the organism or a cell-associated antigen. PCR assays for the diagnosis of CDAD have been reported in the liturature, but these reports are limited in scope and many of the primer pairs used are inefficient or amplify inappropriate portions of the C. difficile genome. We originally selected 5 primer pairs specific for the two toxin genes of C. difficile. We have selected the two primer pairs that are the most efficient in amplifying toxin A and toxin B. The base sequences for these two genes are both very AT rich, and we discovered that this prohibits the development of a sensitive Light Cycler assay. We have developed an ELISA format for our amplicon detection and are now in the process of determining the sensitivity of our assay before using patient specimens.
