probes were designed based on homologous regions of the large subunit ribosomal rna gene the figure this region is highly conserved within all reported species of the enterococcus genus haugland et al 2005 previous researchers frahm and obst 2003 used this region as the priming site for the development of a taqman pcr assay for enterococci detection the primers have been modified to best fit the nasba amplification requirements and molecular beacon design criteria as well as minimize the cross hybridization to each other this primer set has been tested against enterococcus faecalis and e faecium and e avium as well as several other genera of gram positive and gram negative bacteria to determine the specificity of the assay and ensure that no cross reactivity occurs to non target organisms data thus far indicate that the primer set is specific to the genus enterococci the assay sensitivity has been evaluated using serially diluted rna isolated from enterococci cells it is also possible to include cell extract samples from individual species as well as mixtures of all of the enterococci species available because ribosomal rna tends to have a complex secondary structure the nasba assay was initiated by a 65 u00b0 c denaturing step for 3 minutes prior to the 41 u00b0 c amplification step 60 to 90 minutes this initial denaturation is required for some but not all nasba assays and its requirement for efficient amplification of enterococci.