Detection of vancomycin resistant genesvanAandvanBby Cycling Probe Technology

Abstract

Cycling Probe Technology (CPT) has been used to develop gene-based assays for detection of vancomycin resistance genesvanAandvanBin enterococci (VRE). Cycling Probe Technology utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme RNase H when hybridized to its complementary DNA target. Conversion of full-length probe into the cleaved probe fragments is the basis for detection and quantification of the CPT reaction. Two gene-specific probes, each one unique to either thevanAorvanBgene, were utilized for development ofvanAandvanBCPT assays, respectively. BothvanAandvanBCPT assays were used to determine the presence or absence of the corresponding gene in 440 clinical enterococcal isolates. The presence ofvanAandvanBgene sequences was detected in 154 and 131 isolates, respectively. Phenotypic characterization of all isolates was determined through interpretation of conventional susceptibility data obtained with the disk diffusion method. Comparison between disk diffusion characterization and CPT assays revealed 11 discrepant isolates. The identity of these isolates was resolved by polymerase chain reaction (PCR) which confirmed thevanAandvanBCPT assay data. Therefore, compared to conventional phenotyping, both thevanAandvanBCPT assays appeared superior for accurate identification of VanA and VanB isolates.