title: Evaluation of peptide nucleic acid-mediated multiplex real-time PCR kits for rapid detection of carbapenemase genes in gram-negative clinical isolates.
abstract: 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 μL 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.
authors.affiliation: Department of Laboratory Medicine, Kosin University College of Medicine, Busan, Republic of Korea.
authors.forename: Seri
authors.initials: S
authors.lastname: Jeong
authors.affiliation: Department of Laboratory Medicine, Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Jung Ok
authors.initials: JO
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Republic of Korea.
authors.forename: Seok Hoon
authors.initials: SH
authors.lastname: Jeong
authors.affiliation: Department of Dental Hygiene College of Medical and Life Science, Shilla University, Busan, Republic of Korea.
authors.forename: Il Kwon
authors.initials: IK
authors.lastname: Bae
authors.affiliation: Department of Laboratory Medicine, Hallym University College of Medicine, Chuncheon, Republic of Korea. Electronic address: swonkeun@hallym.or.kr.
authors.forename: Wonkeun
authors.initials: W
authors.lastname: Song
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020135:Peptide Nucleic Acids; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D020135:Peptide Nucleic Acids; D001618:beta-Lactamases; C063912:carbapenemase
keywords: Carbapenemase; Gene; Multiplex real-time PCR; Peptide nucleic acid