Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species

•Bacteria from the genus Cronobacter are opportunistic foodborne pathogens.•New primers for PCR-amplification of the rpoB gene were designed.•Three endonucleases were applied for species-specific RFLP patterns analysis.•A simple, cost-effective method is suitable for use in labs with standard equipment.

Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.