BACKGROUND AND OBJECTIVE: Human error and timeless in conventional techniques to identify species Enterococcus faecium and Enterococcus faecalis are two species of the pathogenic species in humans, more accurate techniques is essential. The aim of this study is to design a method of quickly and accurately using DNA melting by Real Time PCR technique to identify and separate the two species in clinical isolates.

METHODS: In this experimental study, the bacterial isolates in the Department of Microbiology Bank of Hamadan University of Medical Sciences was used. Design of primers was done by proprietary software and selecting DivIVA gene for Enterococcus faecalis and alanine racemase for Enterococcus faecium was performed. Isolates identification was evaluated by using Real Time PCR test and melting curve temperature of DNA.

FINDINGS: Susceptibility of primers designed in divIVA gene (specific for E. faecalis) and alanine-racemase gene (specific for E. faecium) was 15CFU/ml per reaction. Specificity of designed primers by using DNA melting curve analysis was 76.6 for E. faecalis and 80.93 for E. faecium which showed considerable different in comparison with another microorganism.

CONCLUSION: Using the results obtained in this study, primers designed were sensitivity and specificity for diagnosis and differentiation of Enterococcus faecium and Enterococcus faecalis species in clinical isolates.