CPT-EIA assays for the detection of vancomycin resistant vanA and vanB genes in enterococci

Cycling Probe Technology (CPT) was combined with a colorimetric enzyme-immuno assay (EIA) to develop two assays for the detection of vanA and vanB genes in vancomycin resistant enterococci (VRE). The CPT-EIA assay employs a gene-specific fluorescein labeled DNA-RNA-DNA probe that gets cleaved within the probe : target duplex. The cleaved DNA probe fragments dissociate from the target, making it available for further cycling. Following the separation of cleaved probe fragments, anti-fluorescein-horseradish peroxidase antibodies are used for the detection of uncleaved probes. The two CPT-EIA assays were used to screen a collection of 440 clinical isolates (Modrusan et al., 1999). All of the 154 VanA and 131 VanB isolates were correctly identified in the vanA and vanB CPT-EIA, respectively. The VanA and VanB isolates were differentiated from vancomycin sensitive enterococci (VSE) and also from the VanC isolates. In addition, an accurate VRE detection in the CPT-EIA assay was shown with cultures grown on eight different media.