Evaluation of peptide nucleic acid-mediated multiplex real-time PCR kits for rapid detection of carbapenemase genes in gram-negative clinical isolates

The emergence of clinical isolates of carbapenemase-producing microbes confers multidrug-resistance to these bacteria and renders them difficult to treat. This study was performed to evaluate peptide nucleic acid (PNA) probe-based multiplex real-time PCR kits used to detect carbapenemase genes. In total, 324 carbapenemase genes, collected from 318 gram-negative clinical isolates in 36 different hospital laboratories, were assayed to evaluate multiplex real-time PCR kits (PANAGENE; Daejeon, Korea). The nine most prevalent carbapenemase genes (KPC, OXA-48, GES, IMP, VIM, NDM, ISAba1-OXA-51, OXA-23, and OXA-58) were included in this study. The sensitivity and specificity of the multiplex real-time PCR assay to all of the carbapenemase genes were above 99.0%, except for ISAba1-OXA-51. The detection limit of the assay was 100 target copies per 25 mu L of reaction volume for all of the nine genetic types of carbapenemases, and-the genes were all detected in a single three-hour PCR. The assay also showed considerable efficiency (above 80.0%), stable reproducibility (coefficient of variation, below 5.0%) and a long shelf-life (more than eight months) with no cross reactivity. The developed PNA-mediated multiplex real-time PCR assay was useful for the rapid, accurate and simultaneous identification of nine carbapenemase genes in gram-negative clinical isolates, suggesting its potential to help choose the appropriate antibiotics and aid the control of carbapenemase genes. (C) 2015 Elsevier B.V. All rights reserved.