RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains

• Main Authors: EMD Scheidegger, SAP Fracalanzza, LM Teixeira, P Cardarelli-Leite
• Format: Article
• Language: English
• Published: Instituto Oswaldo Cruz, Ministério da Saúde 2009-11-01
• Series: Memórias do Instituto Oswaldo Cruz.
• Subjects: 16S rRNA gene PCR-RFLP; Enterococcus identification; food isolates; PCR-based identification; phenotypical characterisation
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762009000700011

Restriction fragment length polymorphism (RFLP) analysis of a PCR-amplified fragment of the 16S rRNA gene was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurised milk and fresh cheese. PCR amplification generated a 275 bp fragment, which was digested with three restriction endonucleases (DdeI, HaeIII, HinfI). The strains were divided into five groups (groups A-E) on the basis of their restriction patterns. Five biochemical tests (arabinose, arginine, manitol, methyl-β-D-glucopyranoside and raffinose) were then performed in addition to RFLP analysis to narrow the identification of enterococcal strains to the species level. PCR-RFLP, in conjunction with the selected biochemical tests, allowed the precise identification of the 21 species of Enterococcus included in the present study. This proposed method is relatively simple and rapid and can be useful as an adjunct tool for accurate identification of Enterococcus.