Optimization of PCR-RFLP Method to Confirm the Identification of Enterococcus faecalis and Enterococcus Faecium Isolated from Food Samples

Abstract

Commercial kits for species identification of Enterococcus faecalis and Enterococcus faecium are in some cases (environmental isolates) incorrect to distinguish this species of enterococci. Also the positive results of multiplex PCR are subjected by amount and purity of genomic DNA. This study aimed to optimize the identification of Enterococcus faecalis and Enterococcus faecium isolated from food samples and from the culture collection by PCR-RFLP. The strains identified by conventional biochemical EN-COCCUS test (reactions to arginine, sorbose, arabinose, manitol, sorbitol, melibiose, raffinose, melezitose) were submitted to multiplex PCR. The PCR amplification of 16S rRNA with universal primers generated a 433 bp product. Restriction fragment length polymorphism (RFLP) was analysed by restriction endonuclease HinfI. The sizes of restriction fragments for Enterococcus faecalis were 285bp, 85bp and 63 bp and for Enterococcus faecium were 370 bp and 63bp.

Keywords

• pcr-rflp
• 16s rrna
• multiplex pcr
• enterococcus faecalis
• enterococcus faecium