High-Resolution Melting Curve Analysis Method: a new method for Rapid Detection of Clinical Enterococcus faecium and Enterococcus Faecalis

Background and objective: High Resolution Melting (HRM) Analysis is a fast, high-throughput method for detection of bacteria. The purpose of this study was to identify Enterococcus species by HRM and evaluate sensitivity and specificity of this method compared with the culture method.Methods: In this study, 49 isolates of Enterococcus were collected from different specimens, which identified using culture and biochemical tests .The divIVA of E. faecalis and the Alanine racemase of E. faecium was used for amplification. After evaluation of sensitivity and specificity of the primers by using HRM based Real time PCR, Enterococcus species were determined based on the temperature range. Finally, melt curve profiles were assessed and analyzed using ABI Step one and HRM Software.Results: Out of 49 Enterococcus.spp, 11 isolates (22.48%) of E.faecalis and 19 isolates (38.77%) of E.faecium were detected. The divIVA gene primers with length of 123bp and Alanine racemase gene primers with length of 248bp were successfully amplified for identification of all the Enterococcus species. Melting temperatures, shown in the melting curves of gene amplification was 79.3±0/5˚C for divIVA gene and 84.5 ± 0/5˚C for Alanine racemase gene. The results revealed that out of 11 clinical isolates of Enterococcus faecium identified by phenotypic method, HRM test was positive in 8 isolates. Also out of the 19 clinical isolates of Enterococcus faecalis, HRM test was positive in 18 isolates.Conclusion: The results of this study showed that the HRM assay was highly reliable, rapid and cost-effective to detect the majority of pathogenic Enterococcus species types.

Keywords:

High-Resolution Melting Curve Analysis (HRM) , Enterococcus faecium , Enterococcus Faecalis