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-immu ne assay (EIA) to develop two assays for the detection of vanA and vanB gen es in vancomycin resistant enterococci (VRE). The CPT-BIA assay employs a g ene-specific fluorescein labeled DNA-RNA-DNA probe that gets cleaved within the probe:target duplex. The cleaved DNA probe fragments dissociate from t he target, making it available for further cycling. Following the separatio n of cleaved probe fragments, anti-fluorescein-horseradish peroxidase antib odies are used for the detection of uncleaved probes. The two CPT-EIA assay s 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 identifi ed in the vanA and vanB CPT-EIA, respectively. The VanA and VanB isolates w ere differentiated from vancomycin sensitive enterococci (VSE) and also fro m the VanC isolates. In addition, an accurate VRE detection in the CPT-EIA assay was shown with cultures grown on eight different media. (C) 2000 Else vier Science Inc. All rights reserved.