PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCI_VC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCI_VC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCI_VC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCI_VM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCI _VM revealed high sequence similarity (96%) with oriCI_VC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCI_VM and oriCI_VC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.