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

Food Microbiol. 2017 Apr:62:1-8. doi: 10.1016/j.fm.2016.08.004. Epub 2016 Aug 26.

Abstract

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.

Keywords: Cronobacter spp.; PCR-RFLP; Species identification; rpoB gene.

MeSH terms

  • Cronobacter / classification*
  • Cronobacter / genetics
  • DNA Primers
  • DNA, Bacterial
  • DNA-Directed RNA Polymerases / genetics*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Food Contamination / analysis
  • Food Microbiology*
  • Genes, Bacterial*
  • Phylogeny
  • Polymerase Chain Reaction / economics
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Restriction Fragment Length*
  • Sequence Analysis, DNA

Substances

  • DNA Primers
  • DNA, Bacterial
  • DNA-Directed RNA Polymerases
  • Deoxyribonucleases, Type II Site-Specific
  • GATC-specific type II deoxyribonucleases
  • GCGC-specific type II deoxyribonucleases
  • GTAC-specific type II deoxyribonucleases