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This study demonstrates a novel detection assay able to identify and subtype strains of Clostridium difficile. Primers carefully designed for melting curve analysis amplify DNA from three C. difficile genes, tcdB, tcdC and cdtB, during quantitative (q)PCR. The tcdB gene allows for confirmation of organism presence, whilst the tcdC and cdtB genes allow for differentiation of virulence status, as deletions in the tcdC gene and the concurrent presence of the cdtB gene, which produces binary toxin, are associated with hypervirulence. Following qPCR, subtyping is then achieved by automated, inline melting curve analysis using only a single intercalating dye and verified by microchip electrophoresis. This assay represents a novel means of distinguishing between toxigenic and hypervirulent C. difficile strains NAP1/027/BI and 078 ribotype, which are highly prevalent hypervirulent strains in humans. This methodology can help rapidly detect and identify C. difficile strains that impose a significant health and economic burden in hospitals and other healthcare settings.

Single fluorophore melting curve analysis for detection of hypervirulent Clostridium difficile