Design and Development of Molecular Beacon-Based Real-Time PCR Assays to Identify Clostridioides difficile Types of Main Evolutionary Clades

C. difficile infection (CDI) has an important impact on both human and animal health. The rapid detection and monitoring of C. difficile PCR-ribotypes (RTs) cause of CDI is critical to control and prevent this infection. This study reports the first application of the Molecular Beacon (MB)-based real-time PCR method in genotyping important C. difficile RTs of the main evolutionary clades. The cdtR gene was used as target and the cdtR sequences were analyzed after extraction from deposited genomes or were obtained after sequencing from strains of different origin. cdtR alleles were identified after sequence comparisons and MB-based real-time PCR assays were developed to discriminate them. In total, 550 cdtR sequences were compared, 38 SNPs were found, and five different cdtR alleles were identified. In total, one or two alleles were associated to the RTs grouped in the same evolutionary clade. A MB-based real-time assay was designed for each allele and for optimized testing of the C. difficile strains. The results obtained demonstrated that the MB-based real-time PCR assays developed in this study represent a powerful, original, and versatile tool to identify C. difficile types/clades and to monitor changes in the population structure of this important pathogen.