Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes

► Multiplex PCR was used to detect C. difficile virulence genes. ► Detection over a wide temperature range enabled sequence analysis. ► Mismatch-tolerant probes were used to distinguish strains. ► Three probes with the same fluorophore identified tcdC sequence variations. ► Signal ratios distinguished variations independent of PCR product concentration.