In Vitro Selection and Characterization of a DNAzyme Probe for Diverse Pathogenic Strains of Clostridium difficile

Clostridium difficile frequently causes an infectious disease known as Clostridium difficile infection (CDI), and there is an urgent need for the development of more effective rapid diagnostic tests for CDI. Previously we have developed an RNA-cleaving fluorogenic DNAzyme (RFD) probe, named RFD-CD1, that is capable of detecting a specific strain of C. difficile but is too specific to recognize other pathogenic C. difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C. difficile but also capable of recognizing diverse pathogenic C. difficile strains. Extensive sequence and structure characterization establishes a pseudoknot structure and a significantly minimized sequence for RFD-CD2. As a fluorescent sensor, RFD-CD2 can detect C. difficile at a concentration as low as 100 CFU/mL, thus making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C. difficile.